Non-invasive, in vitro functional tissue assay systems

ABSTRACT

Provided are functional cell contacts for amplifier and tissue assay systems based on substrate-integrated multifunctional microelectrode arrays implementing stem cell technology. The system covers normal and pathogenic characteristics.

FIELD OF THE INVENTION

The present invention is concerned generally with a specific combinationof substrate-integrated multi-functional electrode arrays and stem celltechnology.

BACKGROUND ART

Precursor cells have become a central interest in medical research. Manytissues in the body have a back-up reservoir of precursors that canreplace cells that are senescent or damaged by injury or disease.Considerable effort has been made recently to isolate precursors of anumber of different tissues for use in regenerative medicine and drugdiscovery. Sources and systems for producing differentiated cells from astem cell population for use wherever a relatively homogenous cellpopulation is desirable have been summarized in for example US patentapplication US2003/0040111. Multi- and pluripotent embryonic stem (ES)cells as well as embryonic germ (EG) cells of the mouse can be inducedto differentiate in culture into a variety of cell types, includingcardiac muscle cells.

Furthermore, ES cell technology is used in toxicity testing. Newchemical compounds are constantly being developed and tested on animals.In addition to industrial and household chemicals, a number of chemicalcompositions are developed each year for use as pharmaceuticals. Rulesregarding the testing of potential pharmaceuticals are promulgated bythe Food and Drug Administration (“FDA”), which currently requirescomprehensive testing of toxicity, mutagenicity, and other effects in atleast two species before a drug candidate can be entered into humanclinical trials. Preclinical toxicity testing alone costs some hundredsof thousands of dollars. Despite this huge investment, almost one thirdof all prospective human therapeutics fail in the first phase of humanclinical trials because of unexpected toxicity. It is clear thatcurrently available toxicological screening assays do not detect alltoxicities associated with human therapy or exposure to chemicals in theenvironment. Better means of screening potential therapeutics orchemicals in general for potential toxicity would reduce the cost anduncertainty of developing new therapeutics and materials, for examplefor use in medical devices or in other devices or goods humans areexposed to every day.

The detection of teratogenic and/or embryotoxic properties of chemicalagents occurs presently by determination of the reproduction toxicity oftest substances following single or multi-administrations to pregnantlaboratory mammals and by tests of the embryotoxicity in the earlystages of pregnancy. Furthermore, in vitro tests are performed withmammal embryos (Neubert and Merker, de Gruyter, Berlin-N.Y. (1981)) andwith embryonic organs for teratogenicity tests. These test procedureshave however the disadvantage that they require the use of a largenumber of live mammals, in particular rats and mice. In vitro testprocedures, in which primary cell cultures (for example, “Limb Buds”,Kochhar, Teratology 11 (1975), 273-287), or brain parts of embryonicrats (Flint and Orton, Toxicol. Appl. Pharmacol. 76 (1984), 383-395) orpermanent cell lines of embryonic or adult mammal tissue, such as tumorcells of the ovary or embryonic palate cells are employed, do notfulfill, strictly speaking, the requirements which are imposed on theteratogenicity tests during the embryogenesis, namely giving indicationsof possible dysgenesis or developmental disturbances.

Efforts have been made for a couple of years to employ cell-based invitro test systems for the detection of toxicity or the efficacy of newpharmaceutical compounds. Those systems depend either on primary cellcultures or on permanent cell lines. Disadvantages of primary cellcultures include laborious preparations, consumption of animals, andvariation between individual animals. Permanent cell lines frequentlyfail to represent physiological conditions.

Hence, there remains always a need for alternative and preferablyimproved assays. For example, U.S. Pat. No. 6,498,018 describes a methodfor determining the effect of a biological agent by contacting a cellculture with a biological agent. The cell culture contains humanmultipotent CNS neural stem cells that are derived from primary CNSneural tissue and a culture medium with preselected growth factors. Theread-out is provided by the effect of a biological agent on the presenceor absence on a biological function or property ascribable to the cellculture. A major disadvantage of this system is the fact that particularbiological functions or properties inherent to a certain culture ofcells are difficult to measure and often involve the destruction of alarge part of the culture in order to obtain enough material for theassay.

WO02/086073 discloses a method for the positive selection of neuronalcells differentiated from nuclear transfer embryonic stem cells bytaking advantage of the neural stem cell marker nectin. This method islimited to neural cell types expressing nectin naturally.

U.S. Pat. No. 6,007,993 describes an in vitro test procedure for thedetection of chemically-induced embryotoxic (for example alsoteratogenic) effects based on differentiated pluripotent embryonic stem(ES) cells from the mouse and rat and using embryonic germ (EG) cellsobtained established from primordial germ cells. Stable transgenic ES orEG stem cell clones are constructed, wherein a bacterial reporter gene,LacZ or the luciferase gene, is brought under the control oftissue-specific promoters or developmental control genes. Followingdifferentiation of the ES cells in the presence of teratogenicsubstances into the different germination path derivatives, there occursa differentiation-dependent expression in the cells, due to the activityof the tissue-specific promoters. The activation, repression ormodulation of these tissue-specific genes is detected based on areaction depending on the reporter gene employed, for example the X-Galassay.

WO99/01552 discloses embryonic stem (ES) cells, which are transfected ina stable manner with a DNA construct encoding a non-cell damagingfluorescent protein and operatively linked thereto a cell- ordevelopment-dependent promoter. Also disclosed is a method fortoxicological monitoring of substances using these ES cell cultures.

Although the above described methods employ semi-quantitative as well asrelatively simple and robust assays, those assays usually are limited byinhomogeneous cell populations and poor detection methods.

Thus, there is a need for cell-based in vitro assay systems that givereliable results. The solution to said technical problem is achieved byproviding the embodiments characterized in the claims, and describedfurther below.

SUMMARY OF THE INVENTION

The present invention relates to a functional cell and tissue assaysystem for identifying, obtaining and/or profiling a compound ofinterest, for example a drug, comprising:

-   -   (a) cultivating a biological material comprising cells, a cell        aggregate, tissue or an organ derived from stem cells,        preferably embryonic stem cells on an electrode array;    -   (b) subjecting said biological material to a test substance; and    -   (c) measuring electrical activity of said biological material        through said electrode array, and optionally analyzing further        parameters.

This method is preferably performed with a multi- or microelectrodearray (MEA). This assay system of the present invention is a particularadvantageous alternative to animal testing for cardiac effect analyses,which are usually quite time-consuming and expensive. Thus, thefunctional tissue assay system is particularly useful in drugdevelopment and toxicity testing of any compound a human or animal mightget in contact with. A particular preferred embodiment of the functionaltissue assay system of the present invention employs so-calledcardiobodies, i.e. embryoid bodies (EBs) differentiated intocardiomyocytes and representing a functional cardiac tissue thatconsists of atrial and ventricular cardiomyocytes as well as ofpacemaker cells.

In particular, the present invention provides a new technology whichresides in the combination of extracellular recording ofelectrophysiological processes via substrate-integrated multifunctionalmicroelectrode arrays (M-MEA) with pH electrodes, pO₂ electrodes andelectrodes for extracellular recording of field potential ofelectrophysiological processes, digital optical analyses and embryonicstem cell (ES cell) technology.

Furthermore, the present invention relates to kits and apparatus usefulfor conducting the method of the present invention, said kits maycomprise vectors or compositions of vectors, arrays, multi- orpluripotent cells, and optionally culture medium, recombinant nucleicacid molecules, standard compounds, etc.

Other embodiments of the invention will be apparent from the descriptionthat follows.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Chip to be used in the assay system of the present invention. Achip to be used in accordance with an assay system of the presentinvention can be derived from commercially available microelectrodearrays such as those described further below and in the examples. Theoptical part of the chip usually comprises a photographic chip, ontowhich the culture areas are built up with the help of light conductorsor optical fibers and which serve for the recording of video sequencesthat can be stored in a computer and analyzed.

DEFINITIONS

For the purposes of this description, the term “stem cell” can refer toeither stem cell or germ cell, for example embryonic stem (ES) and germ(EG), respectively. Minimally, a stem cell has the ability toproliferate and form cells of more than one different phenotype, and isalso capable of self renewal-either as part of the same culture, or whencultured under different conditions. Embryonic stem cells are alsotypically telomerase positive and OCT-4 positive. Telomerase activitycan be determined using TRAP activity assay (Kim et al., Science 266(1997), 2011), using a commercially available kit (TRAPeze® XKTelomerase Detection Kit, Cat. s7707; Intergen Co., Purchase N.Y.; orTeloTAGGG™ Telomerase PCR ELISAplus, Cat. 2,013,89; Roche Diagnostics,Indianapolis). hTERT expression can also be evaluated at the mRNA levelby RT-PCR. The LightCycler TeloTAGGG™ hTERT quantification kit (Cat.3,012,344; Roche Diagnostics) is available commercially for researchpurposes.

In accordance with the present invention, the term embryonic stem (ES)cell includes any multi- or pluripotent stem cell derived frompre-embryonic, embryonic, or fetal tissue at any time afterfertilization, and have the characteristic of being capable underappropriate conditions of producing progeny of several different celltypes that are derivatives of all of the three germinal layers(endoderm, mesoderm, and ectoderm), according to a standard art-acceptedtest, such as the ability to form a teratoma in 8-12 week old SCID mice.“Embryonic germ cells” or “EG cells” are cells derived from primordialgerm cells. The term “embryonic germ cell” is used to describe cells ofthe present invention that exhibit an embryonic pluripotent cellphenotype. The terms “human embryonic germ cell (EG)” or “embryonic germcell” can be used interchangeably herein to describe mammalian,preferably human cells, or cell lines thereof, of the present inventionthat exhibit a pluripotent embryonic stem cell phenotype as definedherein. Thus, EG cells are capable of differentiation into cells ofectodermal, endodermal, and mesodermal germ layers. EG cells can also becharacterized by the presence or absence of markers associated withspecific epitope sites identified by the binding of particularantibodies and the absence of certain markers as identified by the lackof binding of certain antibodies.

“Pluripotent” refers to cells that retain the developmental potential todifferentiate into a wide range of cell lineages including the germline. The terms “embryonic stem cell phenotype” and “embryonic stem-likecell” also are used interchangeably herein to describe cells that areundifferentiated and thus are pluripotent cells and that preferably arecapable of being visually distinguished from other adult cells of thesame animal.

Included in the definition of ES cells are embryonic cells of varioustypes, exemplified by human embryonic stem cells, described by Thomsonet al. (Science 282 (1998), 1145); embryonic stem cells from otherprimates, such as Rhesus stem cells (Thomson et al., Proc. Natl. Acad.Sci. USA 92 (1995), 7844), marmoset stem cells (Thomson et al., Biol.Reprod. 55 (1996), 254) and human embryonic germ (hEG) cells (Shamblottet al., Proc. Natl. Acad. Sci. USA 95 (1998), 13726). Other types ofpluripotent cells are also included in the term. Any cells of mammalorigin that are capable of producing progeny that are derivatives of allthree germinal layers are included, regardless of whether they werederived from embryonic tissue, fetal tissue, or other sources. The stemcells employed in accordance with the present invention that arepreferably (but not always necessarily) karyotypically normal. However,it is preferred not to use ES cells that are derived from a malignantsource.

“Feeder cells” or “feeders” are terms used to describe cells of one typethat are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. The feedercells are optionally from a different species as the cells they aresupporting. For example, certain types of ES cells can be supported byprimary mouse embryonic fibroblasts, immortalized mouse embryonicfibroblasts (such as murine STO cells, e.g., Martin and Evans, Proc.Natl. Acad. Sci USA 72 (1975), 1441-1445), or human fibroblast-likecells differentiated from human ES cells, as described later in thisdisclosure. The term “STO cell” refers to embryonic fibroblast mousecells such as are commercially available and include those deposited asATCC CRL-1503.

The term “embryoid bodies” (EBs) is a term of art synonymous with“aggregate bodies”. The terms refer to aggregates of differentiated andundifferentiated cells that appear when ES cells overgrow in monolayercultures, or are maintained in suspension cultures. Embryoid bodies area mixture of different cell types, typically from several germ layers,distinguishable by morphological criteria; see also infra. As usedherein, “embryoid body”, “EB” or “EB cells” typically refers to amorphological structure comprised of a population of cells, the majorityof which are derived from embryonic stem (ES) cells that have undergonedifferentiation. Under culture conditions suitable for EB formation(e.g., the removal of Leukemia inhibitory factor or other, similarblocking factors), ES cells proliferate and form small mass of cellsthat begin to differentiate. In the first phase of differentiation,usually corresponding to about days 1-4 of differentiation for humans,the small mass of cells forms a layer of endodermal cells on the outerlayer, and is considered a “simple embryoid body”. In the second phase,usually corresponding to about days 3-20 post-differentiation forhumans, “complex embryoid bodies” are formed, which are characterized byextensive differentiation of ectodermal and mesodermal cells andderivative tissues. As used herein, the term “embryoid body” or “EB”encompasses both simple and complex embryoid bodies unless otherwiserequired by context. The determination of when embryoid bodies haveformed in a culture of ES cells is routinely made by persons of skill inthe art by, for example, visual inspection of the morphology. Floatingmasses of about 20 cells or more are considered to be embryoid bodies;see. e.g., Schmitt et al., Genes Dev. 5 (1991), 728-740; Doetschman etal. J. Embryol. Exp. Morph. 87 (1985), 27-45. It is also understood thatthe term “embryoid body”, “EB”, or “EB cells” as used herein encompassesa population of cells, the majority of which being pluripotent cellscapable of developing into different cellular lineages when culturedunder appropriate conditions. As used herein, the term also refers toequivalent structures derived from primordial germ cells, which areprimitive cells extracted from embryonic gonadal regions; see, e.g.,Shamblott, et al. (1998), supra. Primordial germ cells, sometimes alsoreferred to in the art as EG cells or embryonic germ cells, when treatedwith appropriate factors form pluripotent ES cells from which embryoidbodies can be derived; see, e.g., U.S. Pat. No. 5,670,372; Shamblott, etal., supra.

The terms “polynucleotide” and “nucleic acid molecule” refer to apolymer of nucleotides of any length. Included are genes and genefragments, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA and RNA, nucleic acid probes, and primers. As used in thisdisclosure, the term polynucleotides refer interchangeably to double-and single-stranded molecules. Unless otherwise specified or required,any embodiment of the invention that is a polynucleotide encompassesboth a double-stranded form, and each of the two complementarysingle-stranded forms known or predicted to make up the double-strandedform. Included are nucleic acid analogs such as phosporamidates andthiophosporamidates.

A cell is said to be “genetically altered”, “transfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. The genetic alterationis said to be “inheritable” if progeny of the altered cell have the samealteration.

A “regulatory sequence” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, such as replication,duplication, transcription, splicing, polyadenylation, translation, ordegradation of the polynucleotide. Transcriptional control elementsinclude promoters, enhancers, and repressors.

Particular gene sequences referred to as promoters, like the “αMHC” or“collagen” promoter, are polynucleotide sequences derived from the genereferred to that promote transcription of an operatively linked geneexpression product. It is recognized that various portions of theupstream and intron untranslated gene sequence may in some instancescontribute to promoter activity, and that all or any subset of theseportions may be present in the genetically engineered construct referredto. The promoter may be based on the gene sequence of any species havingthe gene; unless explicitly restricted, and may incorporate anyadditions, substitutions or deletions desirable, as long as the abilityto promote transcription in the target tissue. Genetic constructsdesigned for treatment of humans typically comprise a segment that is atleast 90% identical to a promoter sequence of a human gene.

According to the present invention, the term “cell- and/ordevelopment-dependent promoter” is intended to mean a promoter whichdisplays its promoter activity only in particular cell types and/or onlyin particular stages of cellular development, both in cell cultures(embryoid bodies) and in transgenic non-human mammals derived from theES cells according to the invention. In addition, any other knowncell-specific promoter can be employed, e.g. for nerve cells, heartcells, neurons, glia cells, hematopoietic cells, endothelial cells,smooth muscle cells, skeletal muscle cells, cartilage cells, fibroblastsand epithelial cells.

Genetic elements are said to be “operatively linked” if they are in astructural relationship permitting them to operate in a manner accordingto their expected function. For instance, if a promoter helps initiatetranscription of the coding sequence, the coding sequence can bereferred to as operatively linked to (or under control of) the promoter.There may be intervening sequence between the promoter and coding regionso long as this functional relationship is maintained.

In the context of encoding sequences, promoters, and other geneticelements, the term “heterologous” indicates that the element is derivedfrom a genotypically distinct entity from that of the rest of the entityto which it is being compared. For example, a promoter or geneintroduced by genetic engineering techniques into an animal of adifferent species is said to be a heterologous polynucleotide. An“endogenous” genetic element is an element that is in the same place inthe chromosome where it occurs in nature, although other elements may beartificially introduced into a neighboring position.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably in this disclosure to refer to polymers of amino acidsof any length. The polymer may comprise modified amino acids, it may belinear or branched, and it may be interrupted by non-amino acids.

If not stated otherwise the terms “compound”, “substance” and“(chemical) composition” are used interchangeably herein and include butare not limited to therapeutic agents (or potential therapeutic agents),food additives and nutraceuticals, agents of known toxicities such asneurotoxins, hepatic toxins, toxins of hematopoietic cells, myotoxins,carcinogens, teratogens, or toxins to one or more reproductive organs.The chemical compositions can further be agricultural chemicals, such aspesticides, fungicides, nematicides, and fertilizers, cosmetics,including so-called “cosmeceuticals”, industrial wastes or by-products,or environmental contaminants. They can also be animal therapeutics orpotential animal therapeutics. Industrial products that can be testedwith the methods of the present invention include bleaches, toilet,blocks, washing-up liquids, soap powders and liquids, fabricconditioners, window, oven, floor, bathroom, kitchen and carpetcleaners, dishwater detergents and rinse aids, watersoftening agents,descalers, stain removers, polishes, oil products, paints, paintremovers, glues, solvents, varnishes, air fresheners, moth balls andinsecticides.

New ingredients for household products are constantly being developedand needed to be tested. For example, in recent years new enzymes (todigest stains) and “optical brighteners” (which make washing appearwhiter) have been developed for use in washing powders and liquids. Newsurfactants (which cut through grease to remove ingrained dirt) andchemical “builders” (which act as water softeners and enable surfactantsto work more effectively) have been developed for use in washing powdersand liquids, washing-up liquids and various cleaning agents. But alsomedical materials have to be tested, for example dental materials suchas new filling polymers, metal alloys, and bioactive ceramic.Furthermore, chemical compositions of any part of a device, such ascatheters, electrodes, adhesives, paste, gel or cream may be tested withthe method of the present invention in different concentrations and withdifferent ingredients and impurities present.

As used herein, “profile” or “profiling” of a chemical composition orcompound refers to a pattern of alterations in gene or proteinexpression, or both, or physiological properties in an ES cell, embryoidbody, tissue, etc. contacted by the chemical composition compared to alike cell, embryoid body or tissue in contact only with culture medium.

Detailed Description of the Embodiments of the Present Invention

The present invention relates to a functional tissue assay system forobtaining and/or profiling a compound of interest such as a drug,comprising:

-   -   (a) cultivating a biological material comprising cells, a cell        aggregate, tissue or an organ derived from stem cells,        preferably embryonic stem cells on an electrode array;    -   (b) subjecting said biological material to a test substance; and    -   (c) measuring electrical activity of said biological material        through said electrode array, and optionally analyzing further        parameters.

Preferably, the biological material is or is derived from tissue ortissue-like structures obtained by culturing an embryonic stem (ES) cellderived first cell type in the presence of at least one embryonic secondcell type; and allowing integration and alignment of said at least twocell types into tissue or tissue-like structures. A corresponding methodfor providing a variety of tissue or tissue-like structures and likebiological material is described in detail in international applicationWO2004/113515, the disclosure content of which is incorporated herein byreference.

In one embodiment, the biological material is derived from non-humanstem cells.

The invention can be practiced using stem cells of any vertebratespecies. Included are stem cells from humans; as well as non-humanprimates, domestic animals, livestock, and other non-human mammal.Amongst the stem cells suitable for use in this invention are primatepluripotent stem cells derived from tissue formed after gestation, suchas a blastocyst, or fetal or embryonic tissue taken any time duringgestation. Non-limiting examples are primary cultures or establishedlines of embryonic stem cells. The invention is also applicable to adultstem cells. It is referred to the literature of Anderson et al., Nat.Med. 7 (2001), 393-395 and Prockop, Science 276 (1997), 71-74, whereinthe extraction and culture of those cells is described.

Media for isolating and propagating stem cells can have any of severaldifferent formulas, as long as the cells obtained have the desiredcharacteristics, and can be propagated further. Suitable sources includeIscove's modified Dulbecco's medium (IMDM), Gibco, #12440-053;Dulbecco's modified Eagles medium (DMEM), Gibco #11965-092; KnockoutDulbecco's modified Eagles medium (KO DMEM), Gibco #10829-018; 200 mML-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco11140-050; [beta]-mercaptoethanol, Sigma #M7522; human recombinant basicfibroblast growth factor (bFGF), Gibco #13256-029. Exemplaryserum-containing ES medium and conditions for culturing stem cells areknown, and can be optimized appropriately according to the cell type.Media and culture techniques for particular cell types referred to inthe previous section are provided in the references cited herein.

As mentioned before, several sources for ES cells are at the disposal ofthe skilled person of which human stem cells are preferred for most ofthe embodiments of the present invention. Human embryonic stem cells andtheir use for preparing different cell and tissue types are alsodescribed in Reprod. Biomed. Online 4 (2002), 58-63. Embryonic stemcells can be isolated from blastocysts of members of the primate species(Thomson et al., Proc. Natl. Acad. Sci. USA 92 (1995), 7844). HumanEmbryonic Germ (EG) cells can be prepared from primordial germ cellspresent in human fetal material taken about 8-11 weeks after the lastmenstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95 (1998), 13726. Methodfor making cells that resemble embryonic stem cells or embryonic germcells in morphology and pluripotency derived from primordial germ cellsisolated from human embryonic tissue, such as from the gonadal ridges ofhuman embryo, are described in US patent U.S. Pat. No. 6,245,566.

Recently, is has been reported that exfoliated human deciduous tooth, acomparable very accessible tissue, contains multipotent stem cells thatwere identified to be a population of highly proliferative, clonogeniccells capable of differentiating into a variety of cell types includingneural cells, adipocytes, and odontoblasts; see Miura et al., Proc.Natl. Acad. Sci. USA 100 (2003), 5807-5812. After in vivotransplantation, those cells were found to be able to induce boneformation, generate dentin, and survive in mouse brain along withexpression of neural markers. Furthermore, multilineage potential ofhomozygous stem cells derived from metaphase II oocytes has beendescribed by Lin et al. in Stem Cells 21 (2003), 152-161. Varioussources of precursor cells in postnatal muscles and the factors that mayenhance stem cell participation in the formation of new skeletal andcardiac muscle in vivo are reviewed in Grounds et al., J. Histochem.Cytochem. 50 (2002), 589-610. Purification of rare Hematopoietic StemCell(s) (HSC) to homogeneity that home to bone marrow is described inUS2003/0032185. These adult bone marrow cells are described to havetremendous differentiative capacity as they can also differentiate intoepithelial cells of the liver, lung, GI tract, and skin.

The field of stem cell technology is being reviewed by Kiessling andAnderson, Harvard Medical School, in Human Embryonic Stem Cells: AnIntroduction to the Science and Therapeutic Potential; (2003) Jones andBartlett Publishers; ISBN: 076372341X.

Stem cells can be propagated continuously in culture, using acombination of culture conditions that promote proliferation withoutpromoting differentiation. Traditionally, stem cells are cultured on alayer of feeder cells, typically fibroblast type cells, often derivedfrom embryonic or fetal tissue. The cell lines are plated to nearconfluence, usually irradiated to prevent proliferation, and then usedto support when cultured in medium conditioned by certain cells (e.g.Koopman and Cotton, Exp. Cell 154 (1984), 233-242; Smith and Hooper,Devel. Biol. 121 (1987), 1-91), or by the exogenous addition of leukemiainhibitory factor (LIF). Such cells can be grown relatively indefinitelyusing the appropriate culture conditions without differentiation.

In the absence of feeder cells, exogenous leukemia inhibitory factor(LIF), or conditioned medium, ES or EG cells spontaneously differentiateinto a wide variety of cell types, including cells found in each of theendoderm, mesoderm, and ectoderm germ layers. With the appropriatecombinations of growth and differentiation factors, however, celldifferentiation can be controlled. For example, mouse ES and EG cellscan generate cells of the hematopoietic lineage in vitro (Keller et al.,Mol. Cell. Biol. 13 (1993), 473-486; Palacios et al., Proc. Natl. Acad.Sci USA 92 (1995), 7530-7534; Rich, Blood 86 (1995), 463-472).Additionally, mouse ES cells have been used to generate in vitrocultures of neurons (Bain et al., Developmental Biology 168 (1995),342-357; Fraichard et al., J. Cell Science 108 (1995), 3161-3188),cardiomyocytes (heart muscle cells) (Klug et al., Am. J. Physiol. 269(1995), H1913-H1921), skeletal muscle cells (Rohwedel et al., Dev. Biol.164 (1994), 87-101), vascular cells (Wang et al., Development 114(1992), 303-316), U.S. Pat. No. 5,773,255 relates to glucose-responsiveinsulin secreting pancreatic beta cell lines, U.S. Pat. No. 5,789,246relates to hepatocyte precursor cells. Hepatic differentiation of murineembryonic stem cells is also described in Jones et al., Exp. Cell Res.272 (2002), 15-22.

Other progenitors of interest include but are not limited tochondrocytes, osteoblasts, retinal pigment epithelial cells,fibroblasts, skin cells such as keratinocytes, dendritic cells, hairfollicle cells, renal duct epithelial cells, smooth and skeletal musclecells, testicular progenitors, and vascular endothelial cells. Embryonicstem cell differentiation models for cardiogenesis, myogenesis,neurogenesis, epithelial and vascular smooth muscle cell differentiationin vitro have been generally described in Guan et al., Cytotechnology 30(1999), 211-226.

In certain embodiments of the invention, differentiation is promoted bywithdrawing one or more medium component(s) that promote(s) growth ofundifferentiated cells, or act(s) as an inhibitor of differentiation.Examples of such components include certain growth factors, mitogens,leukocyte inhibitory factor (LIF), and basic fibroblast growth factor(bFGF). Differentiation may also be promoted by adding a mediumcomponent that promotes differentiation towards the desired celllineage, or inhibits the growth of cells with undesired characteristics.

In accordance with this invention, populations of differentiated cellsto be used in the assay are preferably depleted of relativelyundifferentiated cells and/or of cells of undesired cell types by usinga selection system that is lethal to the undesired cells and cell types,i.e. by expressing a selectable marker gene that renders cells of aspecific cell type resistant to a lethal effect of an external agent,under control of a regulatory sequence that causes the gene to bepreferentially expressed in the desired cell type and/or at a certainstage of development. To accomplish this, the cells are geneticallyaltered before the process used to differentiate the cells into thedesired lineage, in a way that the cells comprises a selectable markeroperably linked to a first cell type specific regulatory sequencespecific for the desired first cell type.

Any suitable expression vector for this purpose can be used. Suitableviral vector systems for producing stem cells altered according to thisinvention can be prepared using commercially available virus components.The introduction of the vector construct or constructs into theembryonic stem cells occurs in a known manner, e.g. by transfection,electroporation, lipofection or with the help of viral vectors. Viralvectors comprising effector genes are generally described in thepublications referenced in the last section. Alternatively, vectorplasmids can be introduced into cells by electroporation, or usinglipid/DNA complexes. Exemplary is the formulation Lipofectamine 2000™,available from Gibco/Life Technologies. Another exemplary reagent isFuGENE™ 6 Transfection Reagent, a blend of lipids in non-liposomal formand other compounds in 80% ethanol, obtainable from Roche DiagnosticsCorporation. Preferably, the vector constructs and transfection methodsdescribed in WO02/051987 are used; the disclosure content of which isincorporated herein by reference.

Resistance genes per se are known. Examples for these are nucleoside andaminoglycoside-antibiotic-resistance genes, e.g. puromycin(puromycin-N-acetyltransferase), streptomycin, neomycin, gentamycin orhygromycin. Further examples for resistance genes aredehydrofolate-reductase, which confers a resistance against aminopterineand methotrexate, as well as multi drug resistance genes, which confer aresistance against a number of antibiotics, e.g. against vinblastin,doxorubicin and actinomycin D.

In a particularly preferred embodiment of the present invention, saidselectable marker confers resistance to puromycin. Puromycin isparticularly suited for the fast elimination of non-cardiac cells inadherent culture of transgenic EBs. Furthermore, drug selection ofcardiac cells can be implemented entirely in the suspension culture oftransgenic EBs. Hence, it could also be shown that purified ES derivedcardiomyocytes survive much longer in culture than untreatedcounterparts. Moreover, the elimination of undifferentiated ES cellsduring drug selection process has itself been shown to have clearpositive effect on viability and longevity of such differentiated ESderived cells as cardiomyocytes. In addition, it could be surprisinglyshown that the release from surrounding non-differentiated cells inducesproliferation of cardiomyocytes. Thus, the drug selection possesses bothpurifying and multiplying effect.

In a preferred embodiment of the invention, said ES cell of said ES cellderived first cell type comprises a reporter gene, wherein said reporteris operably linked to a cell type specific regulatory sequence specificfor said first cell type. This type of vector has the advantages ofproviding visualization of differentiation, definition of the time pointfor beginning of drug selection, visualization of drug selection andtracing of the fate of purified cells. Such vectors, which arepreferably employed in accordance with the methods of the presentinvention are described in WO02/051987. Usually, said cell type specificregulatory sequence of the reporter gene is substantially the same assaid first cell type specific regulatory sequence of the marker gene.This can advantageously be achieved by putting said marker gene and saidreporter gene into the same recombinant nucleic acid molecule, i.e.vector used for stem cell transfection, preferably such that said markergene and said reporter gene are contained on the same cistron.

The reporter can be of any kind as long as it is non-damaging for thecell and confers an observable or measurable phenotype. According to thepresent invention, the green fluorescent protein (GFP) from thejellyfish Aequorea victoria (described in WO95/07463, WO96/27675 andWO95/21191) and its derivates “Blue GFP” (Heim et al., Curr. Biol. 6(1996), 178-182 and Redshift GFP (Muldoon et al., Biotechniques 22(1997), 162-167) can be used. Particularly preferred is the EnhancedGreen Fluorescent Protein (EGFP). Further embodiments are the EnhancedYelow and Cyan Fluorescent Proteins (EYFP and ECFP, respectively) andRed Fluorescent proteins (DsRed, HcRed). Further fluorescent proteinsare known to the person skilled in the art and can be used according tothe invention as long as they do not damage the cells. The detection offluorescent proteins takes places through per se known fluorescencedetection methods; see, e.g., Kolossov et al., J. Cell Biol. 143 (1998),2045-2056. Alternatively to the fluorescent proteins, particularly in invivo applications, other detectable proteins, particularly epitopes ofthose proteins, can also be used. Also the epitope of proteins, thoughable to damage the cell per se, but whose epitopes do not damage thecells, can be used; see also WO02/051987.

For the selection for stably transfected ES-cells vector constructscontain a further selectable marker gene, which confers e.g. aresistance against an antibiotic, e.g. neomycin; see also supra. Ofcourse, other known resistance genes can be used as well, e.g. theresistance, genes described above in association with the fluorescentprotein encoding genes. The selection gene for the selection for stablytransfected ES-cells is under the control of a different promoter thanthat which regulates the control of the expression of the detectableprotein. Often constitutively active promoters are used, e.g. thePGK-promoter.

The use of a second selection gene is advantageous for the ability toidentify the successfully transfected clones (efficiency is relativelylow) at all. Otherwise a smothering majority of non-transfected ES-cellmay exist and during differentiation e.g. no EGFP positive cells mightbe detected.

In a further embodiment of the invention the cells can be manipulatedadditionally so that specific tissues are not formed. This can occur forinstance by inserting repressor elements, e.g. a doxizyclin induciblerepressor element. Thereby, a possible contamination of the desireddifferentiated cells with pluripotent, potentially tumorigenic cells canbe excluded.

The desired first cell type intended for the stem cell to differentiateto may be of any kind and includes but not limited to neuronal cells,glial cells, cardiomyocytes, glucose-responsive insulin secretingpancreatic beta cells, hepatocytes, astrocytes, oligodendrocytes,chondrocytes, osteoblasts, retinal pigment epithelial cells,fibroblasts, keratinocytes, dendritic cells, hair follicle cells, renalduct epithelial cells, vascular endothelial cells, testicularprogenitors, smooth and skeletal muscle cells; see also supra.

In a particular preferred embodiment of the invention, said first celltype are cardiomyocytes. For this embodiment, said cell type specificregulatory sequence is preferably atrial and/or ventricular specific.Corresponding regulatory sequences, i.e. cardiac specific promoters aredescribed in the prior art; see also supra. For example Nkx-2.5 specificfor very early cardiomyocytes and mesodermal precursor cells,respectively, (Lints et al., Development 119 (1993), 419 431);human-cardiac-α-actin specific for heart tissue, (Sartorelli et al.,Genes Dev. 4 (1990), 1811-1822), and MLC-2V specific for ventricularheart muscle cells (O'Brien et al., Proc. Natl. Acad. Sci. USA. 90(1993), 5157 5161; Lee et al., Mol. Cell Biol. 14 (1994), 1220-1229;Franz et al., Circ Res. 73 (1993), 629-638 and WO96/16163). The cardiacspecific alpha-myosin heavy chain promoter is described in Palermo etal., Cell. Mol. Biol. Res. 41 (1995), 501-519 and Gulick et al., J.Biol. Chem. 266 (1991), 9180-91855. The expression of the atrialspecific myosin heavy chain AMHC1 and the establishment ofanteroposterior polarity in the developing chicken heart is described inYutzey et al., Development 120 (1994), 871-883.

In accordance with this embodiment, it is preferred to use fibroblastsas said at least one embryonic second cell type. Those fibroblasts maynot necessarily be derived from embryos but can also be generated denovo from ES cells in accordance with the method of the presentinvention. Thus, ES cells are transfected with a recombinant nucleicacid molecule comprising a marker and optionally reporter geneoperatively linked to a cell type specific regulatory sequence, i.e.fibroblast specific promoter such as the a2 (I) collagen promoter thoughalso active in bone cells; Lindahl et al., Biol. Chem. 277 (2002),6153-6161; Zheng et al., Am. J. Pathol. 160 (2002), 1609-1617; Antonivet al_, J. Biol. Chem. 276 (2001), 21754-21764; see also Finer et al.,J. Biol. Chem. 262 (1987), 13323-13333; Bou-Gharios et al., J. Cell.Biol. 134 (1996), 1333-1344; Metsaranta et al., J. Biol. Chem. 266(1991) 16862-16869. However, for other embodiments fibroblasts may beused as well and, or alternatively, other supporting cells such asendothelial cells, etc. and derivatives thereof.

In a further preferred embodiment, the biological material employed inthe assay of the present invention is obtained from culturing said atleast two cell types in the presence of an embryonic or embryonic stem(ES) cell derived third cell type. Said third cell type may be any celltype mentioned above. Preferably, said third cell type are endothelialcells. Hence, either embryonic endothelial cells or ES cell derivedendothelial cells may be used. In the latter embodiment, saidendothelial cells are derived from ES cells transfected with a vectorconstruct as generally described before, wherein said cell type specificregulatory sequence is an endothelial specific promoter; see, e.g.,vascular endothelial-cadherin promoter described by Gory et al.,. Blood93(1999), 184-192; the Tie-2 promoter/enhancer by Schlaeger et al.,Proc. Natl. Acad. Sci. USA 94 (1997), 3058-3063; the Flk-1promoter/enhancer by Kappel et al., Biochem. Biophys. Res. Commun. 276(2000), 1089-1099.

Further cell and tissue type specific promoters are known; see, e.g.,chondrocyte specific pro-alphal (II) collagen chain (collagen 2)promoter fragment described by Zhou et al., J. Cell Sci. 108 (1995),3677-3684; neural alpha-1-tubulin specific promoter described in Glosteret al., J Neurosci 14 (1994); 7319-7330 and glial fibrillary acidicprotein (GFAP) promoter in Besnard et al., J. Biol. Chem. 266 (1991);18877-18883. Furthermore, see, e.g., Kawai et al., Biochim. Biophys.Acta 1625 (2003), 246-252 and Kugler et al., Gene Ther. 10 (2003),337-347 for glial and neuronal specific promoters. Efficiency ofembryoid body formation and hematopoietic development from embryonicstem cells in different culture systems is described for example in Danget al., Biotechnol. Bioeng. 78 (2002), 442-453.

“Tissue specific” is to be subsumed under the term “cell specific”.

Further examples for non-heart specific promoters are: PECAM1, FLK-1(endothelium), nestine (neuronal precursor cells),tyrosin-hydroxylase-1-promoter (dopaminergic neurons), smooth musclea-actin, smooth muscle myosin (smooth muscles), α1-fetoprotein(endoderm), smooth muscle heavy chain (SMHC minimal promoter (specificfor smooth muscles, (Kallmeier et al., J. Biol. Chem. 270 (1995),30949-30957).

The term development specific promoter refers to promoters that areactive during certain points of time during development. Examples forsuch promoters are the β-MHC promoter that is expressed during embryonaldevelopment in the ventriculum of the mouse and is superseded by theα-MHC promoter in the prenatal phase; NK×2.5, a promoter during theearly mesoderm/heart development; atrial-natriuretic-factor, a marker ofthe early embryonal heart with exception of the pacemaker, that is downregulated also in later developmental stages; Flk-1, an endotheliumspecific promoter that is active during the early vasculogenesis; intron2-segment of the nestine gene that is expressed in neuronal precursorcells (embryonal neurons and glia cells) and adult glia cells (partiallystill able to divide) (Lothian and Lendahl, Eur. J. Neurosci. 9 (1997),452-462U).

For the embodiments described hereinbefore, said resistance gene andsaid reporter gene are preferably contained in a bicistronic vector andare preferably separated by an IRES. Particular preferred is the use ofa construct, wherein said resistance gene confers resistance topuromycin, said marker is EGFP and said promoter is the cardiac αMHCpromoter.

As mentioned before, in accordance with the present invention any ofsaid at least two cell types such as a main cell type and correspondingsupporting cells may be derived from ES cells and used in the assay ofthe present invention. Thus, tissue or tissue-like structures to beassayed in accordance with present invention can be obtained by a methodcomprising the following steps:

-   -   (a) transfecting one or more multi- or pluripotent cells with        recombinant nucleic acid molecules comprising a first and a        second cell type specific regulatory sequence operably linked to        at least one selectable marker, wherein said second cell type is        different from said first cell type;    -   (b) culturing the cells under conditions allowing        differentiation of the cells; and    -   (c) isolating cells of at least two differentiated cell types        and/or eliminating non-differentiated cells, optionally along        with cells differentiating towards irrelevant cell types from        cell types of interest that activate the selectable marker in        the course of differentiation.

Similarly as in the previous methods the generation of more than twocell types is desired. Therefore, the method preferably comprisestransfecting said one or more cells with recombinant nucleic acidmolecules comprising at least one further cell type specific regulatorysequence operably linked to at least one selectable marker, wherein saidat least one further cell type is different from said first and secondcell type. For use in the method, said recombinant nucleic acidmolecules are comprised in the same vector or different vectors.

The cell type may be selected from the group consisting of neuronalcells, glial cells, cardiomyocytes, glucose-responsive insulin secretingpancreatic beta cells, hepatocytes, astrocytes, oligodendrocytes,chondrocytes, osteoblasts, retinal pigment epithelial cells,fibroblasts, keratinocytes, dendritic cells, hair follicle cells, renalduct epithelial cells, vascular endothelial cells, testicularprogenitors, smooth and skeletal muscle cells; see also supra.

Promoters that are preferably used if the preparation of cardiac tissueis desired by differentiating the transfected stem cell(s) intocardiomyocytes, fibroblasts and optionally endothelial cells comprisethose described hereinbefore. Similarly, for producing neuronal tissueone or more stem cells, for example multipotent neural stem cells, canbe used and genetically engineered in accordance with the presentinvention to differentiate into neurons, astrocytes, andoligodendrocytes. The same rationale applies for the generation of forexample liver or pancreatic tissue. Regulatory sequences ofcorresponding cell type specific promoters can be obtained from theliterature; see, e.g., “medline” and NCBI.

The above mentioned method can be performed in different ways. First, aspreferably described herein, a multiple transgenic ES clone is producedstably transfected with a certain number of vectors with a drugselection cassette driven by specific promoters accordingly to the celltypes constituting the desirable tissue type. Thus, at least one of saidES cells or cell clone thereof is transfected and selected, wherein saidcell or cell clone contains recombinant nucleic acid molecules with atleast two different cell type specific regulatory sequences. In such avariant all emerging cell types have the origin from one common ES cellclone predecessor and the resulting ratio between different cellcomponents depends on relative differentiation rate of each of them.

Alternatively, at least two different ES cells or clones thereof aretransfected and selected, wherein said at least two different cells orcell clones contain recombinant nucleic acid molecules with differentcell type specific regulatory sequences. By this approach a number oftransgenic ES clones is generated where each single clone possess onlyone vector with drug resistant cassette driven by one of the cell typespecific promoters.

For tissue modeling the relevant clones should be mixed on initial phaseof differentiation (“hanging drops” or “mass culture”) in order to formES cell aggregates (EBs) where, after drug selection, emerging celltypes have origin from different corresponding ES cell clones and thefinal ratio of the cell components also depends on and can be controlledby initial ratio between different ES cell lines. This method preferablyresults in cell aggregates that are chimeric embryoid bodies (EBs).

Irrespective of the particular embodiment of the assay of the invention,it is preferred that in the cells constituting the biological materialto be tested at least two of said selectable markers are operably linkedto said different cell type specific regulatory sequences are identical.As mentioned before, those marker or marker genes are preferablyselectable markers which confer resistance to a cell toxic agent,preferably puromycin, methothrexate, or neomycin.

As described hereinbefore, said one or more of said recombinant nucleicacid molecules may further comprise a reporter operably linked to saidcell type specific sequence; see supra. Herein preferred as well are thedifferent color versions of Enhanced Green Fluorescent Protein (EGFP),in particular EYFP (yellow), ECFP (blue) and/or hcRFP (red) operablylinked to different cell type specific sequences. Likewise preferred isthat said selectable marker and said reporter are expressed from abicistronic vector, preferably wherein said selectable marker and saidreporter are separated one or more Internal Ribosomal Entry Sites(IRES), which are operably linked to at least one of said genes.

As mentioned above, the biological material to be tested is obtained bya method which is preferably performed such that it allows self-assemblyof the different cell types, for example into the desired tissue ortissue-like structures that should reflect the tissue or organ of amammal, preferably human, that is supposed to be exposed to a givencompound. The stem cells are in a preferred embodiment of the inventionavailable in form of aggregates that are known as embryoid bodies (EBs).WO02/051987 describes a protocol to obtain embryoid bodies. Themanufacturing takes place preferably with the “hanging drop” method orby methylcellulose culture (Wobus et al., Differentiation 48 (1991),172-182).

Alternatively to this, spinner flasks (stirring cultures) can be used asculture method. To this end, the undifferentiated ES-cells areintroduced into stirring cultures and are mixed permanently according toan established procedure. Therefore, 10 million ES-cells are introducedinto 150 ml medium with 20% FCS and are stirred constantly with the rateof 20 rpm., wherein the direction of the stirring motion is changedregularly. 24 hours after introduction of the ES-cells an extra 100 mlmedium with serum is added and thereupon 100-150 ml of the medium isexchanged every day (Wartenberg et al., FASEB J. 15 (2001), 995-1005).Under these culture conditions large amounts of ES-cell-derived cells,i.e. cardiomyocytes, endothelial cells, neurons etc. depending on thecomposition of the medium can be obtained. The cells are selected bymeans of the resistance gene either still within the stirring culture orafter plating, respectively.

Alternatively to this, the EBs differentiated in the hanging drop mightbe not plated, but kept simply in suspension. Even under theseconditions a progression of a differentiation could be observedexperimentally. The washing off of the non desired cell types can bedone with mechanical mixing alone and addition of low concentration ofenzyme (e.g. collagenase, trypsin); a single cell suspension is achievedwith easy washing off of the non desired cell types.

In a particular preferred embodiment of the present invention, embryoidbodies are prepared according a recent developed “mass culture” systememployed in the appended examples and described in detail ininternational application WO2005/005621.

Hence, in a particular preferred embodiment, the functional tissue assayof the present invention is performed with embryoid bodies (EBs),preferably chimeric EBs.

As mentioned before, embryoid bodies represent a complex group of cellsdifferentiating into different tissues. In one embodiment, the cellswithin an embryoid body are substantially synchronized for theirdifferentiation. Accordingly, at known intervals, the majority of thesynchronized cells differentiate into the three embryonic germ layersand further differentiate into multiple tissue types, such as cartilage,bone, smooth and striated muscle, and neural tissue, including embryonicganglia; see also Snodgrass et al., “Embryonic Stem Cells: Research andClinical Potentials” in Smith and Sacher, eds. Peripheral Blood StemCells American Association of Blood Banks, Bethesda Md. (1993). Thus,the cells within embryoid bodies provide a much closer model to thecomplexity of whole organisms than do traditional single cell or yeastassays, while still avoiding the cost and difficulties associated withthe use of mice and larger mammals. Moreover, the recent availability ofhuman embryoid bodies improves the predictive abilities of the inventionby providing an even closer vehicle for modeling toxicity andidentification of drugs useful for the treatment of heart disorders inhuman organ systems, and in humans.

The embryoid body to be used in the assay of the invention preferablycomprises a cell population, the majority of which being pluripotentcells capable of developing into different cellular lineages whencultured under appropriate conditions. It is preferred that the embryoidbody comprises at least 51% pluripotent cells derived from totipotent EScells. More preferably, the embryoid body comprises at least 75%pluripotent cells derived from totipotent ES cells. And still morepreferably, the embryoid body comprises at least 95% pluripotent cellsderived from totipotent ES cells.

In its simplest form, the assay of the present invention comprisescreating a molecular profile involving contacting embryoid bodies on anelectrode array with a chemical composition of interest, and thendetermining the alterations electrical activity of said biologicalmaterial through said electrode array, and optionally analyzing furtherparameters such as those described below in the embryoid body exposed tothe chemical composition (the “test embryoid body”) compared to aembryoid body which was not exposed to the agent (a “control embryoidbody”).

The assay of the present invention can also be performed such that itallows self-assembly of the cells into the aggregates or tissue-likestructures onto the array. Alternatively, the differentiated cells aredissociated from the EBs and are analyzed onto the array in cellsuspension or at a single cell level; see also the Examples. Hence, inone preferred embodiment, the cells to be analyzed in the functionalassay system of the present invention are obtained by dissociation fromembryoid bodies (EBs), preferably by trypsinization of the cellaggregates; see also the examples.

In a particularly preferred embodiment, the method of the presentinvention is performed with stem cell-derived EGFP-positivecardiomyocytes, preferably ventricular-like cardiomyocytes or atrial andpacemaker-like cardiomyocytes; see also the examples.

In one embodiment, the fate of the cell types and formation of cellaggregates and tissue as well physiological and/or developmental statusof the cells or cell aggregate are analyzed, before, during and/or afterbeing exposed to the test compound, for example by isometric tensionmeasurements, echocardiography and the like; see also infra. Preferably,the status of the cells or cell aggregates is analyzed by monitoring thedifferentiation of electrical activity of the cells on an array, forexample by recording the extracellular field potentials with amicroelectrode array (MEA). Microelectrode arrays (MEAs) are deviceswhich allow the multiple extracellular recording of action potentialgeneration and propagation within for example ES cell derivedcardiomyocytes. These recordings resemble the well-known ECG as it isused by physicians. The matrix of the MEAs usually consists of 60 goldelectrodes integrated into the bottom of a specially designed cellculture device. ES cell derived embryoid bodies (EBs) can be cultured insuch devices. After attachment and spreading on the surface, the cellsof the EBs containing the cardiomyocytes get in contact with theelectrodes. All outcoming extracellular action potentials can then berecorded synchronously during both short- and long time observationexperiments. The following analysis of frequencies and latencies with anappropriate program allows to reveal the fine “electrical map” of thebeating clusters.

For example, electrophysiological properties during the ongoingdifferentiation process of embryonic stem cells differentiating intocardiac myocytes can be followed by recordings of extracellular fieldpotentials with a microelectrode array (MEA) consisting of 60substrate-integrated electrodes has been described in Banach et al., Am.J. Physiol. Heart Circ. Physiol. 284 (2003), 2114-2123. Furthermore,Multiple arrays of tungsten microelectrodes were used to record theconcurrent responses of brain stem neurons that contribute torespiratory motor pattern generation; see Morris et al., Respir.Physiol. 121 (2000), 119-133.

In a further preferred embodiment, embryoid bodies or selected,dissociated and replated in vitro differentiated cells are cultured andanalyzed on fibronectin-coated cell culture dishes. In a particularlypreferred embodiment, the micro-electrode arrays to be used inaccordance with the assay system of the present invention are coatedwith fibronectin; see also Example 2.

This embodiment is particularly advantageous since it could be shownthat on the one hand fibronectin coating allowed the constant discretelocation of the seeded cells and on the other hand did not interferewith growth and viability of the cells. Methods of coating surfaceswith, for example, fibronectin are known to the person skilled in theart; see also the User Manual for the Micro-Electode Array (MEA) ofMultichannel Systems.

In a particularly preferred embodiment of the functional assay system ofthe present invention, the parameters frequency, mean contractility,maximum contraction amplitude and the mean area under the curve (AUC) ofcontractility are analyzed. Without intending to be bound by theory itis believed in accordance with the present invention that the parametersmean contractility and maximum contraction (Amplitude) reflect thephysiological effects of for example β-adrenergic receptor agonists andcalcium antagonists, i.e. increase and decrease of contractility andfrequency, respectively, in a correct manner, while correspondingvehicle controls should not show any change.

Any substance may be tested for the above-mentioned parameters,optionally in comparison with known agonists and antagonists,respectively. From the resultant values for the parameters for examplemedium contractility and maximum contraction amplitude it is thenpossible to qualitatively and quantitatively characterize the testsubstance, for example, whether it is an agonist or antagonist, andwhether its effect is more or less pronounced than a given standard. Themethod of the present invention is thus perfectly suitable for toxicitytesting and profiling of compounds.

Methods for computational analysis of microarray data are known in theart; see, e.g., Quackenbush, Nature Reviews 2 (2001), 418-427 and Brazmaet al., Nature Genetics 29(2001), 365-371. Generally, statisticalanalysis can be done using the 2-tailed t test and consideringdifferences to be significant at P<0.05 or P<0.01. Linear regressionanalysis can be performed and correlation coefficients can be determinedusing the program Excel 2000 (Microsoft Seattle, Wash., USA). Clusteranalysis on microarray data can be performed by using for exampleprograms available as shareware from Michael Eisen's laboratory(http://rana.lbl.gov).

In one preferred embodiment of the method of the present invention, EScell derived in vitro differentiated cardiomyocytes are enzymaticallydissociated from cell aggregates such as EBs by trypsinization.Cardiomyocytes, preferably of a particular type, e.g. atrial andpacemaker or ventricular are selected for contractile analysis. Thecardiomyocytes are loaded onto a MEA, preferably coated withfibronectin, mounted on the stage of a Zeiss Axiovert 25 (20× Objective)inverted microscope preferably through a heating adapter andcontinuously superfused with oxygenated physiological buffer, forexample containing 132 mM NaCl, 4.8 mM KCl, 12 mM MgCl₂, 10 mM HEPES, 5mM pyruvic acid, 1.8 mM CaCl₂, pH 7.2, at 37° C. The cardiomyocytes canbe paced using a Myopacer field stimulator (IonOptix, Milton, Mass.) toproduce contraction at 1, 2, 5, and 10 Hz in the absence or presence offor example 10 nM isoproterenol. An IonOptix video system (Milton,Mass.) can be used to record the cell length by two-edge detection. Datacan be acquired at a sampling rate of 240 Hz and analyzed by theSoftEdge computer program from IonOptix. The data can be exported to theprogram Excel, and statistic Analysis can be carried out using Student ttest.

Hence, the assay of the invention can be used for a variety of purposes,for example for analyzing the influence of factors and compounds ontissue formation during embryonic development.

In a further embodiment, the present invention relates to arrays for usein the assay of the present invention comprising a solid support andattached thereto or suspended thereon cells, cell aggregates or tissueobtained by the method of the present invention or being in thedifferentiation process. The use of planar microelectrode arrays forcultured cells and cell aggregates as biosensors is of particularinterest. Such arrays generally consist of a substrate of glass, plasticor silicon over which a conductor, e.g. gold, platinum,indium-tin-oxide, iridium, etc., is deposited and patterned. Aninsulating layer, e.g. photoresist, polyimide, silicon dioxide, siliconnitride, etc., is deposited over the conducting electrodes andinterconnects and then removed in regions over the electrodes to definethe recording sites. Cells are cultured directly on this surface andcontact the exposed conductor at the deinsulated recording sites.Depending on the size of the electrodes and the cells, recordings ofelectrical activity can be from a single cell or populations of cellsincluding cell aggregates. Each electrode site is generally connected tothe input of a high input impedance, low noise amplifier, with orwithout AC coupling capacitors, to allow amplification of the relativelysmall extracellular signals. Examples of such biosensors are describedby Novak et al. IEEE Transactions on Biomedical Engineering BME-33(2)(1986), 196-202; Drodge et al., J. Neuroscience Methods 6 (1986),1583-1592; Eggers et al., Vac. Sci. Technol. B8(6) (1990), 1392-1398;Martinoia et al., J. Neuroscience Methods 48 (1993), 115-121; Maeda etal., J. Neuroscience 15 (1995), 6834-6845; and Mohr et al. Sensors andActuators B-Chemical 34 (1996), 265-269.

An apparatus prepared and adapted for analyzing the above describedarrays is also subject of the present invention.

The functional tissue assay system of the present invention isparticularly suited for use in drug screening and therapeuticapplications. For example, differentiated stem cells can be used toscreen for factors (such as solvents, small molecule drugs, peptides,polynucleotides, and the like) or environmental conditions (such asculture conditions or manipulation) that affect the characteristics ofdifferentiated cells. Particular screening applications of thisinvention relate to the testing of pharmaceutical compounds in drugresearch. The reader is referred generally to the standard textbook “Invitro Methods in Pharmaceutical Research”, Academic Press, 1997, andU.S. Pat. No. 5,030,015). Assessment of the activity of candidatepharmaceutical compounds generally involves combining the differentiatedcells of this invention with the candidate compound, determining anychange in the morphology, marker phenotype, or metabolic activity of thecells that is attributable to the compound (compared with untreatedcells or cells treated with an inert compound), and then correlating theeffect of the compound with the observed change. The screening may bedone, for example, either because the compound is designed to have apharmacological effect on certain cell types, or because a compounddesigned to have effects elsewhere may have unintended side effects. Twoor more drugs can be tested in combination (by combining with the cellseither simultaneously or sequentially), to detect possible drug-druginteraction effects. In some applications, compounds are screenedinitially for potential toxicity (Castell et al., pp. 375-410 in “Invitro Methods in Pharmaceutical Research,” Academic Press, 1997).Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and expression or release ofcertain markers, receptors or enzymes. Effects of a drug on chromosomalDNA can be determined by measuring DNA synthesis or repair. [H]thymidineor BrdU incorporation, especially at unscheduled times in the cellcycle, or above the level required for cell replication, is consistentwith a drug effect. Unwanted effects can also include unusual rates ofsister chromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

The above mentioned parameters may be used in the functional tissueassay system of the present invention as any one of said furtherparameters besides the measuring of electrical activity of saidbiological material through said electrode array.

Preferably, embryoid bodies are used in the assays of the presentinvention to test the chemical composition; see also infra. The choiceof the particular species from which the embryoid body is derived willtypically reflect a balance of several factors. First, depending on thepurpose of the study, one or more species may be of particular interest.For example, human embryoid bodies will be of particular interest foruse with compositions being tested as potential human therapeutics butalso for toxicological tests for substances including industrialchemicals, while equine, feline, bovine, porcine, caprine, canine, orsheep embryoid bodies may be of more interest for a potential veterinarytherapeutic. Embryoid bodies of other species commonly used inpreclinical testing, such as guinea pigs, mice, rat, rabbits, pigs, anddogs, are also preferred. Typically, embryoid bodies of these specieswill be used for “first pass” screening, or where detailed informationon toxicity in humans is not needed, or where a result in a murine orother one of these laboratory species has been correlated to a knowntoxicity or other effect in humans. Furthermore, with respect to humantherapeutics, regulatory agencies generally require animal data beforehuman trials can begin; it will generally be desirable to use embryoidbodies of species which will be used in the preclinical animal studies.The results of toxicity testing in the embryoid bodies can then guidethe researcher on the degree and type of toxicity to anticipate duringthe animal trials. Certain animal species are known in the art to bebetter models of human toxicity of different types than are others, andspecies also differ in their ability to metabolize drugs; see, e.g.,Williams, Environ. Health Perspect. 22 (1978), 133-138; Duncan, Adv.Sci. 23 (1967), 537-541. Thus, the particular species preferred for usein a particular preclinical toxicity study may vary according to theintended use of the drug candidate. For example, a species which providea suitable model for a drug intended to affect the reproductive systemmay not be as suitable a model for a drug intended to affect the nervoussystem. Criteria for selecting appropriate species for preclinicaltesting are well known in the art.

Once an embryoid body culture has been initiated, it can be contactedwith a chemical composition. Conveniently, the chemical composition isin an aqueous solution, preferably in a solvent conventionally used incell culture, for example DMSO, and is introduced to the culture medium;see also the Examples. The introduction can be by any convenient means,but will usually be by means of a pipette, a micropipettor, or asyringe. In some applications, such as high throughput screening, thechemical compositions will be introduced by automated means, such asautomated pipetting systems, which may be on robotic arms. Chemicalcompositions can also be introduced into the medium as in powder orsolid forms, with or without pharmaceutical excipients, binders, andother materials commonly used in pharmaceutical compositions, or withother carriers which might be employed in the intended use. For example,chemical compositions intended for use as agricultural chemicals or aspetrochemical agents can be introduced into the medium by themselves totest the toxicity of those chemicals or agents, or introduced incombination with other materials with which they might be used or whichmight be found in the environment, to determine if the combination ofthe chemicals or agents has a synergistic effect. Typically, thecultures will be shaken at least briefly after introduction of achemical composition to ensure the composition is dispersed throughoutthe medium.

The time as which a chemical composition is added to the culture iswithin the discretion of the practitioner and will vary with theparticular study objective. Conveniently, the chemical composition willbe added as soon as the embryoid body develops from the stem cells,permitting the determination of the alteration in protein or geneexpression on the development of all the tissues of the embryoid body.It may be of interest, however, to focus the study on the effect of thecomposition on a particular tissue type. As previously noted, individualtissues, such as muscle, nervous, and hepatic tissue, are known todevelop at specific times after the embryoid body has formed. Additionof the chemical composition can therefore be staged to occur at the timethe tissue of interest commences developing, or at a chosen time aftercommencement of that development, in order to observe the effect onaltering gene or protein expression in the tissue of interest.

Different amounts of a chemical composition will be used to contact anembryoid body or cell depending on the amount of information known aboutthe toxicity of that composition, the purposes of the study, the timeavailable, and the resources of the practitioner. A chemical compositioncan be administered at just one concentration, particularly where otherstudies or past work or field experience with the compound haveindicated that a particular concentration is the one which is mostcommonly found in the body. More commonly, the chemical composition willbe added in different concentrations to cultures of embryoid bodies orcells run in parallel, so that the effects of the concentrationdifferences on gene or protein expression and, hence, the differences intoxicity of the composition at different concentrations, can beassessed. Typically, for example, the chemical composition will be addedat a normal or medium concentration, and bracketed by twofold orfivefold increases and decreases in concentration, depending on thedegree of precision desired.

Where the composition is one of unknown toxicity, a preliminary study isconveniently first performed to determine the concentration ranges atwhich the composition will be tested. A variety of procedures fordetermining concentration dosages are known in the art. One commonprocedure, for example, is to determine the dosage at which the agent isdirectly toxic. The practitioner then reduces the dose by one half andperforms a dosing study, typically by administering the agent ofinterest at fivefold or twofold dilutions of concentration to parallelcultures of cells of the type of interest. For environmentalcontaminants, the composition will usually also be tested at theconcentration at which it is found in the environment. For agriculturalchemicals, such as pesticides which leave residues on foodstuffs, theagent will usually be tested at the concentration at which the residueis found, although it will likely be tested at other concentrations aswell. Thus, the dilution of test compounds can be done by making inseparated tubes a series of dilution of 50 or 100 fold concentratedcompounds in DMSO. One or two μl of each dilution are distributed ineach well before cell suspension distribution.

The above considerations with respect to contacting the compounds withthe EBs, contacting time, etc., also apply to the assays of theinvention performed on e.g. ES cells, tissue and non-human animals, ifapplicable.

Hence, as mentioned above the present invention relates to a functionaltissue assay system for identification, obtaining and/or profiling adrug, comprising:

-   -   (a) cultivating a biological material comprising cells, a cell        aggregate, tissue or an organ prepared in accordance with the        present invention on an electrode array;    -   (b) subjecting said biological material to a test substance; and    -   (c) measuring electrical activity of said biological material        through said electrode array, and optionally further parameters.

This assay is preferably performed with a multi- or microelectrode array(MEA), such as those mentioned above. This assay system of the presentinvention is a particular advantageous alternative to animal testing forcardiac affect analyses, which are usually quite time-consuming andexpensive. Thus, the functional tissue assay system is particularlyuseful in drug development and toxicity testing of any compound a humanor animal might get in contact with. Preferably, said cell aggregatesare EBs; see also supra.

A particular preferred embodiment of the functional tissue assay systemof the present invention employs so-called cardiobodies, i.e. embryoidbodies (EBs) differentiated into cardiomyocytes and representing afunctional cardiac tissue that consists of atrial and ventricularcardiomyocytes as well as of pacemaker cells. Usually said electrodearray is a multi- or microelectrode array (MEA) such as those describedhereinabove.

In accordance with the assay system of the present invention, preferablyany one or all of the following parameters are analyzed:

-   -   (i) Na⁺ channels;    -   (ii) Ca²⁺/K⁺ channels;    -   (iii) K⁺ channels;    -   (iv) Amplitude and/or Field potential duration (FDP),    -   (v) Chronotrophy of cardiac cells or burst periods of neuronal        cells;    -   (vi) Arrhythmias, EAD like phenomena;    -   (vii) pH-value;    -   (viii) oxygen partial pressure (pO2);    -   (ix) Beating arrest; and    -   (x) Analysis of AV-Dissociation contractility, NO-effects and/or        morphological changes.

MEAS and methods for their use in analyses of biological cells are knownto the person skilled in the art. For example, international applicationWO97/05922 describes a microelectrode arrangement for leaking, withlocal resolution, electrical cell potentials, or for electricalstimulation of networks of biological cells such as for example cellcultures, tissue slices “in vitro” or biological tissue “in vivo”. Amicro-element device such as described in international applicationWO98/22819 may be used, which has a plurality of microelements, whichmay be configured as microelectrodes, arranged on a substrate andadapted for making contact to cells present in a liquid environment. Thecells are guided onto the microelectrodes, are isolated or aremechanically attracted to the microelectrodes. A negative-pressure forceor a hydrodynamic force may be applied on the cells. In addition, theuse of an electrode array as described in international applicationWO01/65251 may be adapted in accordance with the teaching of the presentinvention.

For analyses of the multielectrode data several tools available in theprior art may be used, see for example Egert et al., “MEA-tools: An opensource toolbox for the analysis of multielectrode data with MATLAB. J.Neuroscience Methods 117 (2002), 33-42, and Banach et al., Am. J.Physiol. Heart Circ. Physiol. 284 (2003), H2114-2123).

In a preferred embodiment, the biological material comprises embryoidbodies (EBs) differentiated into cardiomyocytes, most preferably EBsthat consist of functional cardiac tissue that beats autonomously andcovers electrophysiological properties of atrial and ventricularcardiomyocytes, as well as of pacemaker cells.

The methods and assays described herein can replace various animalmodels, and form novel human based tests and extreme environmentbiosensors. In particular, the methods of the invention can be used fortoxicological, mutagenic, and/or teratogenic in vitro tests. Since thecells and tissue obtained in accordance with the present invention moreclosely resemble the in vivo situation the results obtained by thetoxicological assays of the present invention are expected to correlateto in vivo teratogenicity of the tested compounds as well.

In a particular advantageous embodiment of the present invention, theabove described assays are used as a system alternative for animaltesting of cardiac effects of compounds, which is quite time consumingand expensive. This embodiment is based on “cardiobodies”, i.e. embryoidbodies (EBs) differentiated into cardiomyocytes, preferably thoseobtainable by the method described in international applicationWO2005/005621. Said cardiobodies are preferably derived from mouseembryonic stem cells and consist of functional cardiac tissue that beatsautonomously and covers electrophysiological properties of atrial andventricular cardiomyocytes, as well as of pacemaker cells.

In a particular preferred embodiment, ES cells of the mouse cell line R1(Nagy et al., Proc. Natl. Acad. Sci. 90 (1993), 8424-8428; ATCC NumberSCRC-1011) or a cell line derived thereof are used in the assays of thepresent invention. Experiments performed in accordance with the presentinvention revealed that the use of cardiomyocytes derived from themurine ES cell line R1 led to a substantial improvement of the signalnoise ratio of the multielectrode array system of the present inventioncompared to the use of cardiomyocytes derived from the D3 cell line(Doetschman et al., J. Embryol. Exp. Morphol. 87 (1985), 27-45; Proc.Natl. Acad. Sci. USA 85 (1988), 8583-8587; ATCC Number CRL-1934 andCRL-11632). Without intending to be bound by theory it is believed thatthe improvement of the assay is due to a better adhesion of thisparticular cell line on the multielectrode array.

In one embodiment, cardiobodies or cells dissociated therefrom areplated on a multielectrode array system (MEA, MultiChannel Systems,Reutlingen, Germany). Recordings of extracellular field potentials withmicroelectrode arrays consisting of 60 substrate-integrated electrodescan be done as described for example in Banach et al., Am. J. Physiol.Heart Circ. Physiol. 284 (2003), H2114-2123. Extracellular recordings ofthe field potential reflect the electrophysiological changes duringexcitation of the cardiomyocytes in cardiobodies. In a particularpreferred embodiment, automated analysis is performed using theAxioTools software developed by the Axiogenesis AG, Cologne, Germany.

The assay of the present invention was evaluated with compounds withknown effects on ion channels present in cardiac tissue, for examplecisapride, lidocaine, isoproterenol and nifedipine, which were testedeither in the assay of the present invention or in rabbit Langendorffpreparations. All compounds showed similar results in both test systemsas well as a correlation in Patch Clamp and MEA analysis, which leads tothe implementation of the assay of the present invention in the cardiacsafety screening routine. In order to achieve most reliable results,preferably most if not all of the following parameters are analyzed, ifapplicable:

-   -   (ii) Na⁺ channels;    -   (ii) Ca²⁺/K⁺ channels;    -   (iii) K⁺ channels;    -   (iv) Amplitude and/or Field potential duration (FDP),    -   (v) Chronotrophy of cardiac cells or burst periods of neuronal        cells;    -   (vi) Arrhythmias, EAD like phenomena;    -   (vii) pH-value;    -   (viii) oxygen partial pressure (pO2);    -   (ix) Beating arrest; and    -   (x) Analysis of AV-Dissociation contractility, NO-effects and/or        morphological changes.

The advantages of this particular embodiment of screening assays of thepresent invention over conventional in-vitro assays include

-   -   highly standardized cell culture model, homogeneous and        reproducible production of cardiobodies;    -   presence of atrial, ventricular, and pacemaker cells with normal        physiological behavior (e.g. expression and regulation of ion        channels);    -   ECG-like screening of all electrophysiological properties of the        cardiobody including effects on all ion channels, chronotropy        and appearance of arrhythmias;    -   entirely in vitro-based system, no requirement for laborious        cell preparation    -   time- and cost-saving

Thus, in the various assays of the present invention compounds, inparticular cardiac active compounds can be tested in accordance withmethods described in DE 195 25 285 A1; Seiler et al., ALTEX 19 Suppl 1(2002), 55-63; Takahashi et al., Circulation 107 (2003), 1912-1916 andSchmidt et al., Int. J. Dev. Biol. 4-5 (2001), 421-429; the latterdescribing ES cell test (EST) used in a European Union validation studyfor screening of embryotoxic agents by determiningconcentration-dependently the differentiation of ES cells into cardiacand myogenic cells.

Cells and tissue of the central nervous system (CNS) generated by themethods of the present invention or during differentiation is saidmethods can be tested, for example, in cell culture such as described inU.S. Pat. No. 6,498,018. Similarly, cells and tissue related to theliver can be tested; see, e.g., US2003/0003573. A further in vitro testprocedure for the detection of chemically induced effects on embryonicdevelopment and for differentiation for the purpose ofembryotoxicity/teratogenicity screening based on differentiatedpluripotent embryonic stem (ES) cells from mice and rats using embryonicgerm (EG) cells obtained from primordial germ cells is described inWO97/01644 and can be adapted in accordance with teachings of thepresent invention.

Cells and tissue of the CNS may also be analyzed using an electrodearray as described above. Means and methods for analyzing regulatoryinteractions of neuronal activity of cells and tissue cultures onmicroelectrode arrays are known to the person skilled in the art; seefor example van Bergen et al., Brain Res. Brain Res. Protocol 2003/11(2003), 123-133 and international application WO01/65251.

Preferred compound formulations for testing do not include additionalcomponents, such as preservatives, that have a significant effect on theoverall formulation; see also supra. Thus preferred formulations consistessentially of a biologically active compound and a physiologicallyacceptable carrier, e.g. water, ethanol, DMSO, etc. However, if acompound is liquid without an excipient the formulation may consistessentially of the compound itself Furthermore, a plurality of assaysmay be run in parallel with different compound concentrations to obtaina differential response to the various concentrations. As known in theart, determining the effective concentration of a compound typicallyuses a range of concentrations resulting from 1:10, or other log scale,dilutions. The concentrations may be further refined with a secondseries of dilutions, if necessary. Typically, one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection.

Compounds of interest encompass numerous chemical classes, thoughtypically they are organic molecules; see also supra. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Compounds and candidate agents are obtained from a wide variety ofsources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds and biomolecules, includingexpression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced. Forexample, inhibition of tumor-induced angiogenesis andmatrix-metalloproteinase expression in confrontation cultures ofembryoid bodies and tumor spheroids by plant ingredients used intraditional chinese medicine has been described by Wartenberg et al. inLab. Invest. 83 (2003), 87-98.

Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

The compounds may also be included in a sample including fluids to whichadditional components have been added, for example components thataffect the ionic strength, pH, total protein concentration, etc. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g. under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1 μ1to 1 ml of a biological sample is sufficient.

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. While many sampleswill comprise compounds in solution, solid samples that can be dissolvedin a suitable solvent may also be assayed. Samples of interest includeenvironmental samples, e.g. ground water, sea water, mining waste, etc.;biological samples, e.g. lysates prepared from crops, tissue samples,etc.; manufacturing samples, e.g. time course during preparation ofpharmaceuticals; as well as libraries of compounds prepared foranalysis; and the like. Samples of interest compounds being assessed forpotential therapeutic value, i.e. drug candidates.

The test compound may optionally be a combinatorial library forscreening a plurality of compounds. Such a collection of test substancescan have a diversity of about 10³ to about 10⁵ is successively reducedin running the method, optionally combined with others twice or more.Compounds identified in the method of the invention can be furtherevaluated, detected, cloned, sequenced, and the like, either in solutionor after binding to a solid support, by any method usually applied tothe detection of a specific DNA sequence such as PCR, oligomerrestriction (Saiki et al., Bio/Technology, 3 (1985), 1008-1012),allele-specific oligonucleotide (ASO) probe analysis (Conner et al.,Proc. Natl. Acad. Sci. USA, 80 (1983), 278), oligonucleotide ligationassays (OLAs) (Landegren et al., Science, 241 (1988), 1077), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren et al., Science, 242 (1988), 229-237). Hence, the method ofthe present invention can also be used for transcriptional profiling ofembryonic and adult stem cells; see, e.g., Ramalho-Santos et al.,Science 298 (2002), 597-600; Tanaka et al., Genome Res. 12 (2002),1921-1928.

Incubating includes conditions which allow contact between the testcompound and the ES cell or ES derived cells. As described above, it isbe desirable to test an array of compounds or small molecules on asingle or few ES cells on a “chip” or other solid support. For example,cardiomyocytes or neurons on chips would give a readout of the rate ofcontraction or number of firings, respectively, in response to acompound and for the detection of harmful or at least biologicallyactive environmental agents.

Neuronal biologically compatible electrode arrays allow the stem cellsto undergo further differentiation on the array itself. These arraysallow the measurement of real time changes in electrical activity in theES derived neurons in response to the presence of known or unidentifiedagents. The electrical activity of cardiomyocytes can be monitored byplating the cells on an array of extracellular microelectrodes (Connollyet al., Biosens. Biores. 5 (1990), 223-234). The cells show regularcontractions, and the extracellular signal recorded showed arelationship to intracellular voltage recordings (Connolly et al.,supra). This noninvasive method allows long term monitoring and issimpler and more robust than typical whole cell patch clamp techniques.

The assay of the present invention is particularly suited to providemodulation reference patterns and databases of modulation referencepatterns for a wide range of biologically active compounds. Thereference patterns are then used for the identification andclassification of test compounds. Evaluation of test compounds may beused to achieve different results. Methods for the classification ofbiological agents according to the spectral density signature of evokedchanges in cellular electric potential are known to the person skilledin the art; see, e.g., U.S. Pat. No. 6,377,057. Thus, biologicallyactive compounds are classified according to their effect on ionchannels, changes in membrane potential and ionic currents, and thefrequency content of action potentials that the compound(s) evoke inexcitable cells. The spectral density changes of such evoked membranepotential or action potential are a characteristic for each channel typethat is modulated by the test compound. A pattern of spectral changes inmembrane potential is determined by contacting a responsive cell with acompound, and monitoring the membrane potential or ionic currents overtime. These changes correlate with the effect of that compound, or classof compounds, on the ion channels of the responding cell. This patternof spectral changes provides a unique signature for the compound, andprovides a useful method for characterization of channel modulatingagents. The effect of a compound on ion channels, and on the actionpotential of a living cell, can provide useful information about theclassification and identity of the compound. Methods and means forextracting such information are of particular interest for the analysisof biologically active compounds, with specific applications inpharmaceutical screening, drug discovery, environmental monitoring,biowarfare detection and classification, and the like. Examples of wholecell based biosensors are described in Gross et al., Biosensors andBioelectronics 10 (1995), 553-567; Hickman et al. Abstracts of PapersAmerican Chemical Society 207 (1994), BTEC 76; and Israel et al.American Journal of Physiology: Heart and Circulatory Physiology 27(1990), H1906-H1917.

Connolly et al., Biosens. Biores. 5 (1990), 223-234 describe a planararray of microelectrodes developed for monitoring the electricalactivity of cells in culture. The device allows the incorporation ofsurface topographical features in an insulating layer above theelectrodes. Semiconductor technology is employed for the fabrication ofthe gold electrodes and for the deposition and patterning of aninsulating layer of silicon nitride. The electrodes were tested using acardiac cell culture of chick embryo myocytes, and the physical beatingof the cultured cells correlated with the simultaneous extracellularvoltage measurements obtained. The molecular control of cardiac ionchannels is reviewed by Clapham, Heart Vessels Suppl. 12 (1997),168-169. Oberg and Samuelsson, J. Electrocardiol. 14 (1981), 13942,perform fourier analysis on the repolarization phases of cardiac actionpotentials. Rasmussen et al. American Journal of Physiology 259 (1990),H370-H389, describe a mathematical model of electrophysiologicalactivity in bullfrog atria.

A large body of literature exists in the general area of ion channels. Areview of the literature may be found in the series of books, “The IonChannel Factsbook”, volumes 1-4, by Edward C. Conley and William J.Brammar, Academic Press. An overview is provided of: extracellularligand-gated ion channels (ISBN: 0121844501), intracellular ligand-gatedchannels (ISBN: 012184451X), inward rectifier and intercellular channels(ISBN: 0121844528), and voltage gated channels (ISBN: 0121844536).Hille, B. (1992) “Ionic Channels of Excitable Membranes”, 2.sup.nd Ed.Sunderland Mass.: Sinauer Associates, also reviews potassium channels.

In another aspect, the biological material is screened for bioactivesubstances. In on example, the cells are coupled with a substrate suchthat electrophysiological changes in flue cells in response to externalstimuli can be measured, e.g., for use as a high-throughput screen forbioactive substances. The cells can also be transfected with DNA thattargets, expresses, or knocks-out specific genes or gene products in thecell. By providing such chip-mounted cells coupled with measuringdevices, such as a computer, many compounds can be screened rapidly andaccurately. The cells or chips could also be coupled to the measuringdevice in arrays for large-scale parallel screening.

The assay methods of the present invention can be in conventionallaboratory format or adapted for high throughput. The term “highthroughput” (HTS) refers to an assay design that allows easy analysis ofmultiple samples simultaneously, and capacity fox robotic manipulation.Another desired feature of high throughput assays is an assay designthat is optimized to reduce reagent usage, or minimize the number ofmanipulations in order to achieve the analysis desired.

In another preferred embodiment, the method of the present inventioncomprises taking 2, 3, 4, 5, 7, 10 or more measurements, optionally atdifferent positions within the array. In one embodiment of the screeningmethods of the present invention a compound known to activate or inhibitdifferentiation process and/or tissue structure formation is added tothe sample or culture medium, for example retinoic acid; for appropriatecompounds see also supra.

Furthermore, the above-described methods can, of course, be combinedwith one or more steps of any of the above-described screening methodsor other screening methods well known in the art. Methods for clinicalcompound discovery comprises for example ultrahigh-throughput screening(Sundberg, Curr. Opin. Biotechnol. 11 (2000), 47-53) for leadidentification, and structure-based drug design (Verlinde and Hol,Structure 2 (1994), 577-587) and combinatorial chemistry (Salemme etal., Structure 15 (1997), 319-324) for lead optimization. Once a drughas been selected, the method can have the additional step of repeatingthe method used to perform rational drug design using the modified drugand to assess whether said modified drug displays better affinityaccording to for example interaction/energy analysis. The method of thepresent invention may be repeated one or more times such that thediversity of said collection of compounds is successively reduced.

Substances are metabolized after their in vivo administration in orderto be eliminated either by excretion or by metabolism to one or moreactive or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24(1996), 449-459). Thus, rather than using the actual compound or drugidentified and obtained in accordance with the methods of the presentinvention a corresponding formulation as a pro-drug can be used which isconverted into its active form in the patient by his/her metabolism.Precautionary measures that may be taken for the application ofpro-drugs and drugs are described in the literature; see, for review,Ozama, J Toxicol. Sci. 21 (1996), 323-329.

Furthermore, the present invention relates to the use of a compoundidentified, isolated and/or produced by any of these methods for thepreparation of a composition for the treatment of disorders related to,for example damaged tissue or aberrant tissue or organ formation, heartinsufficiency, etc.; see also supra. Preferably, the isolated compoundor corresponding drug supports wound healing and/or healing of damagedtissue. As a method for treatment the identified substance or thecomposition containing it can be administered to a subject sufferingfrom such a disorder. Compounds identified, isolated and/or produced bythe method described above can also be used as lead compounds in drugdiscovery and preparation of drugs or prodrugs. This usually involvesmodifying the lead compound or a derivative thereof or an isolatedcompound as described hereinbefore such as modifying said substance toalter, eliminate and/or derivatize a portion thereof suspected causingtoxicity, increasing bioavailability, solubility and/or half-life. Themethod may further further comprise mixing the substance isolated ormodified with a pharmaceutically acceptable carrier. The various stepsrecited above are generally known in the art. For example, computerprograms for implementing these techniques are available; e.g., Rein,Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss,New York, 1989). Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, New York, USA. Furthermore, peptidomimeticsand/or computer aided design of appropriate derivatives and analoguescan be used, for example, according to the methods described above.Methods for the lead generation in drug discovery also include usingproteins and detection methods such as mass spectrometry (Cheng et al.,J. Am. Chem. Soc. 117 (1995), 8859-8860) and some nuclear magneticresonance (NMR) methods (Fejzo et al., Chem. Biol. 6 (1999), 755-769;Lin et al., J. Org. Chem. 62 (1997), 8930-8931). They may also includeor rely on quantitative structure-action relationship (QSAR) analyses(Kubinyi, J. Med. Chem. 41 (1993), 2553-2564, Kubinyi, Pharm. UnsererZeit 23 (1994), 281-290) combinatorial biochemistry, classical chemistryand others (see, for example, Holzgrabe and Bechtold, Pharm. Acta Hely.74 (2000), 149-155). Furthermore, examples of carriers and methods offormulation may be found in Remington's Pharmaceutical Sciences.

Once a drug has been selected in accordance with any one of theabove-described methods of the present invention, the drug or a pro-drugthereof can be synthesized in a therapeutically effective amount. Asused herein, the term “therapeutically effective amount” means the totalamount of the drug or pro-drug that is sufficient to show a meaningfulpatient benefit, i.e., treatment, healing, prevention or amelioration ofdamaged tissue, or an increase in rate of treatment, healing, preventionor amelioration of such conditions. In addition or alternatively, inparticular with respect to pre-clinical testing of the drug the term“therapeutically effective amount” includes the total amount of the drugor pro-drug that is sufficient to elicit a physiological response in anon-human animal test.

In one embodiment, the method of the invention further comprises mixingthe substance isolated or modified with a pharmaceutically acceptablecarrier. Examples of carriers and methods of formulation may be found inRemington's Pharmaceutical Sciences.

As mentioned above, the methods and assays of the present invention canbe used for toxicological, embryotoxic, mutagenic, and/or teratogenic invitro tests; see supra. Another important aspect of the presentinvention is therefore a method of determining toxicity, preferablyteratogenicity, embryotoxicity, chronic or acute toxicity of a compoundcomprising the steps of the methods described herein.

The assays may be simple “yes/no” assays to determine whether there is aresponsive change compared to a control. The test compound or aplurality of test compounds can also be subjected to the test cell,preferably embryoid body in different concentrations or dilution series,preferably at doses that correspond to physiological levels of thecorresponding type of test compounds. It is thus also possible to easygenerate compound profiles in purpose similar to those described inWO00/34525. For example, two or more assays may be used and/orparameters may be assessed. Those assays/parameters can beperformed/assessed in parallel or subsequently; or the results of oneassay may be compared with results of a corresponding assay performedelsewhere. Thus, a molecular profile of a test chemical composition canbe established by detecting the alterations in the “electrical activity”in, for example, embryoid bodies contacted by the test chemicalcomposition as described in previous sections. Once the molecularprofile of the test composition is determined, it can be compared tothat of a chemical composition with predetermined toxicities or,preferably, to a library of molecular profiles of chemical compositionswith predetermined toxicities. The outcome of such comparison provideinformation for one to predict the likelihood of whether the testcomposition is toxic, what type of toxicities, and how toxic it would beas compared to the other known toxic compositions.

For the purpose of practicing the invention, the predictions of toxicityof the test composition based on its molecular profiles in ES cells,tissue, etc., preferably EB cells does not have to be 100% accurate. Tohave a major positive impact on the efficiency and costs of drugdevelopment, one only has to modestly increase the probability that theless toxic and thus more successful drug candidates are, for example, onthe top half of a prioritized list of new drug leads.

Since the cells and tissue obtained in accordance with the presentinvention more closely resemble the in vivo situation compared toconventional cell based assays, the results obtained by the assays ofthe present invention are expected to correlate to in vivoteratogenicity or embryotoxicity of the tested compounds as well.

Several test substances can be combined and either added simultaneouslyor sequentially to gain information about possible enhancing orquenching effects. Thus a further aspect of the invention relates to themethod described previously, wherein said contacting step furtherincludes contacting said test sample with at least one second testsubstance in the presence of said first test substance. Two or moresubstances tested in combination will provide information about theirinteraction in general.

A preferred embodiment of the methods according to the present inventioninvolves adding a compound known to activate or inhibit differentiationprocess to the culture medium, particularly if this test substance is atherapeutic agent or a mixture thereof. This screening may be done, forexample, either because the compound is known to have a effect on thedifferentiation of certain cell types and is tested to determine itspotential for guiding the differentiation of other cell types, orbecause a compound designed to have effects elsewhere may haveunintended side effects. The last aspect applies particularly totherapeutic agents.

The present invention also relates to kit compositions containingspecific reagents such as those described herein-before useful forconducting any one of the above described methods of the presentinvention, containing the vector or the composition of vectors describedhereinbefore, multi- or pluripotent cells, and optionally culturemedium, recombinant nucleic acid molecules, standard compounds, etc.Such a kit would typically comprise a compartmentalised carrier suitableto hold in close confinement at least one container. The carrier wouldfurther comprise reagents useful for performing said methods. Thecarrier may also contain a means for detection such as labeled enzymesubstrates or the like.

Furthermore, the present invention relates to a chip comprising anelectrode array as defined hereinabove. In a particularly preferredembodiment, the present invention relates to arrays and chips comprisinga solid support and attached thereto or suspended thereon cells, cellaggregates or tissue obtained by the method of the present invention orbeing in the differentiation process. Such arrays generally consist of asubstrate of glass, plastic or silicon on which the test cellsaggregates or tissues are deposited in a particular pattern. Dependingon the type of array, these might additionally be covered by aconductor, e.g. gold, platinum, indium-tin-oxide, iridium, etc., whichallows a direct measurement by employing the conductivity of cells. Theuse of such planar microelectrode arrays for cultured cells and cellaggregates as biosensors is of particular interest.

Preferably, the chip of the present invention is characterized by thepresence of embryoid bodies or cardiomyocytes obtainable by the methodsof the present invention described herein and illustrated in theExamples. In a particular preferred embodiment, the chip of the presentinvention comprises cardiobodies, i.e. cardiac-like tissues includingatrial and ventricular cardiomyocytes as well as pacemaker cells. In aparticular preferred embodiment, said chip comprises a surface asillustrated in FIG. 1.

In addition, the present invention relates to an apparatus for use inthe methods and assays of the present invention described herein. Forexample, a cell-potential measurement apparatus having a plurality ofmicroelectrodes and which may be used and/or adapted in accordance withthe teaching of the present invention is described in European patentapplication EP 0 689 051 A3.

Furthermore, international application WO98/54294 describes an apparatusand method for monitoring cells and a method for monitoring changes incells upon addition of an analyte to the cell's environment, comprisinga device which includes an array of microelectrodes disposed in a cellculture chamber, upon which array a portion of cells adhere to thesurfaces of the microelectrodes. The diameter of the cells are largerthan the diameters of the microelectrodes. A voltage signal is appliedacross each of the microelectrodes and a reference electrode. Detectionand monitoring of the signals resulting from the application of thevoltage signal provides information regarding the electricalcharacteristics of the individual cells, including impedance (combinedcell membrane capacitance and conductance), action potential parameters,cell membrane capacitance, cell membrane conductance, and cell/substrateseal resistance.

Further means and methods that may be implemented in accordance with theteaching of the present invention can be found in the literature, seefor example Egert et al., Brain Res. Brain Res. Protoc. 2 (1998),229-242; Duport et al., Biosens. Bioelectron. 14 (1999), 369-376 andGerman patent application DE 195 29 371 A1.

Hence, the means and methods of the present invention describedherein-before can be used in a variety of applications including but notlimited to “loss of function” assays with ES cells containing homozygousmutations of specific genes, “gain of function” assays with ES cellsoverexpressing exogenous genes, developmental analysis ofteratogenic/embryotoxic compounds in vitro, pharmacological assays andthe establishment of model systems for pathological cell functions, andapplication of differentiation and growth factors for induction ofselectively differentiated cells which, can be used as a source fortissue grafts; see for review, e.g., Guan et al., Altex 16 (1999),135-141.

Yet another aspect of the present invention relates to a method ofconducting a drug discovery business, comprising:

-   -   providing one or more assay systems or components thereof as        described herein for identifying a drug candidate; and/or    -   conducting therapeutic profiling of drugs identified in the        previous step, or further analogs thereof, for efficacy and        toxicity according to the assays of the present invention; and    -   formulating a pharmaceutical preparation including one or more        drugs identified in the previous step as having an acceptable        therapeutic profile.

Utilizing the methods described above, the identity of a drug isdetermined. Agents are identified by their ability to alter the certainparameters such as those described hereinbefore, e.g. those describedfor MEAS. For suitable lead compounds that are identified, furthertherapeutic profiling of the agent, or analogs thereof, can be carriedout for assessing efficacy and toxicity in animals. Those compoundshaving therapeutic profiles after animal testing can be formulated intopharmaceutical preparations for use in humans or for veterinary uses.The subject business method can include an additional step ofestablishing a distribution system for distributing the pharmaceuticalpreparation for sale, and may optionally include establishing a salesgroup for marketing the pharmaceutical preparation.

Instead of developing the identified drug in house, further drugdevelopment can also be achieved by a different company. Thus a furtheraspect of the present invention relates to a method of conducting atarget discovery business comprising:

-   -   providing one or more assay systems described herein or        components thereof for identifying a drug;    -   alternatively or in addition conducting therapeutic profiling of        drugs for efficacy and toxicity according to the assays of the        present invention; and    -   licensing, to a third party, the rights for further drug        development and/or sales for drugs identified or profiled, or        analogs thereof.

For suitable lead compounds that have been identified, further profilingof the agent, or further analogs thereof, can be carried out forassessing efficacy and toxicity in animals, depending on the modalitiesof the agreement with the respective third party. Further development ofthose compounds for use in humans or for veterinary uses will then beconducted by the third party. The subject business method will usuallyinvolve either the sale or licensing of the rights to develop saidcompound but may also be conducted as a service, offered to drugdeveloping companies for a fee.

The present invention also relates to drugs identified according to themethods and assays described above as well as to pharmaceuticalcompositions for use in therapy comprising such a drug.

The drug according to the invention can be combined with suitablediluents or carriers, preferably those which are pharmaceuticallyacceptable. Examples of such carriers, diluents and methods offormulation may be found in Remington's Pharmaceutical Sciences. To forma pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe modulator. Carriers or diluents are usually sterile and non-toxic,and defined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like. A therapeutically effective dose refers tothat amount of modulator which is sufficient to achieve the desiredeffect on differentiation of target cells.

Further examples of suitable pharmaceutical carriers are well known inthe art and include phosphate buffered saline solutions, emulsions, suchas oil/water emulsions, various types of wetting agents; sterilesolutions etc. Compositions comprising such carriers can be formulatedby well known conventional methods. Accordingly, the present inventionalso provides a method of making a pharmaceutical composition for use inmodulating cell differentiation comprising mixing a modulator of celldifferentiation identified according to a method of the invention with asuitable diluent or carrier.

The above disclosure generally describes the present invention. A morecomplete under-standing can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples and figure which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention. The contents of all cited references (including literaturereferences, issued patents, published patent applications as citedthroughout this application and manufacturer's specifications,instructions, etc.) are hereby expressly incorporated by reference;however, there is no admission that any document cited is indeed priorart as to the present invention.

The practice of the present invention will employ, unless otherwiseindicated; conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. For furtherelaboration of general techniques concerning stem cell technology, thepractitioner can refer to standard textbooks and reviews, for exampleTeratocarcinomas and embryonic stem cells: A practical approach (E. J.Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in MouseDevelopment (P. M. Wasserman et al., eds., Academic Press 1993);Embryonic Stem Cell Differentiation in Vitro (Wiles, Meth. Enzymol. 225(1993), 900,); Properties and uses of Embryonic Stem Cells: Prospectsfor Application to Human Biology and Gene Therapy (Rathjen et al.,Reprod. Fertil. Dev. 10 (1998), 31,). Differentiation of stem cells isreviewed in Robertson, Meth. Cell Biol. 75 (1997), 173; and Pedersen,Reprod. Fertil. Dev. 10 (1998), 31. Besides the sources for stem cellsdescribed already above further references are provided; see Evans andKaufman, Nature 292 (1981), 154-156; Handyside et al., Roux's Arch. Dev.Biol., 196 (1987), 185-190; Flechon et al., J. Reprod. Fertil. AbstractSeries 6 (1990), 25; Doetschman et al., Dev. Biol. 127 (1988), 224-227;Evans et al., Theriogenology 33 (1990), 125-128; Notarianni et al., J.Reprod. Fertil. Suppl., 43 (1991), 255-260; Giles et al., Biol. Reprod.44 (Suppl. 1) (1991), 57; Strelchenko et al., Theriogenology 35 (1991),274; Sukoyan et al., Mol. Reprod. Dev. 93 (1992), 418-431; Iannaccone etal., Dev. Biol. 163 (1994), 288-292. Methods in molecular genetics andgenetic engineering are described generally in the current editions ofMolecular Cloning: A Laboratory Manual, (Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press); DNA Cloning, Volumes I and II (D. N. Glover ed.,1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Gene Transfer Vectorsfor Mammalian Cells (Miller & Calos, eds.); Current Protocols inMolecular Biology and Short Protocols in Molecular Biology, 3rd Edition(F. M. Ausubel et al., eds.); and Recombinant DNA Methodology (R. Wued., Academic Press). Gene Transfer Vectors For Mammalian Cells (J. H.Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory);Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, and ClonTech.General techniques in cell culture and media collection are outlined inLarge Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8(1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73);Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991),375); and Suspension Culture of Mammalian Cells (Birch et al.,Bioprocess Technol. 19 (1990), 251). Other observations about the mediaand their impact on the culture environment have been made by MarshallMcLuhan and Fred Allen.

EXAMPLES Example 1 Differentiation Protocol for ES Cells for thePreparation of Ventricular Cardiomyocytes and MEA Recording

For differentiation of ES cells into ventricular cardiomyocytes ES cellsof cell line R1 (see supra and, e.g., Nagy et al., Proc. Natl. Acad.Sci. 90 (1993), 8424-8428) were cultured on 10 cm Petri dishes inDulbecco's-modified Eagle's medium (DMEM) supplemented with 15% fetalcalf serum (FCS) and leukemia inhibitory factor (LIF) on a layer offeeder cells (irradiated mouse embryonic fibroblasts). Cells wereincubated at 37° C., 7% CO₂ and 95% humidity.

-   -   Day 0: Cells were trypsinized to a single cell suspension and        collected by centrifugation. Cells were resuspended to a density        of approximately 2×10⁶ cells per ml in Knock-Out (KO) medium        supplemented with 15% serum replacement (SR). 4 ml per 6 cm        Petri dish of this suspension were incubated on a rocking table        at 50 rpm, 37° C., 5% CO₂ and 95% humidity for the following        day;    -   Day 1: Dilution of the ES cell aggregates (embryonic bodies;        EBs) in KO medium supplemented with 15% SR to a density of 2000        EBs/10 ml and further incubation;    -   Day—3: Change of medium (MW) with KO medium supplemented with        15% SR;    -   Day 4: MW onto Iscove's medium supplemented with 20% FCS;    -   Day 5: MW with Iscove's medium supplemented with 20% FCS;    -   Day 6: MW onto KO medium supplemented with 15% SR;    -   Day 7: For use of the EBs in the MEA assay of the invention, EBs        were plated in 100μl Iscove's medium supplemented with 15% FCS        with 1 EB per culture area and sealed with a polydimethylsilane        (PDMS) closure and used for the assay from Day 10 onwards.

For the preparation of cardiac tissue for the assay recombinant EScells, which express the puromycin resistance gene under the control ofthe heart-specific promoters (alpha MHC for atrial and pacemaker cellsor RLC-2v for ventricular cells), were subjected on Day 9 to 2 μgpuromycin/ml and incubated for further three days. After dissociation ofthe cells with collagenase on Day 12 cardiac cells were mixed withembryonic fibroblasts in a ratio of 1:1 and seeded on the multielectrodearray with a density of 3×10⁵ cells per cm². After the following threedays a cardiac-like tissue develops, which can be analyzed in themultielectrode assay of the invention. For example,substrate-integrated, planar MEAs (Multi Channel Systems, Reutlingen,Germany) for long-term recordings of the spontaneous electrical activityfrom cultures of cardiac myocytes and EBs can be used; see also Egert etal., (1998) and Banach et al., (2003), and references cited therein. EBscan be positioned in the middle of a sterilized MEA consisting of 60Titanium Nitride coated gold electrodes (∅=30 μm; inter-electrodedistance 200 μm in a square grid). For recording, a separate sterileAg/AgCl electrode can be temporarily inserted into the dish as groundelectrode. The MEA can be connected to the amplifier and dataacquisition system (Multi Channel System, Reutlingen, Germany), whichincludes a heating device to maintain a constant temperature of 37° C.Data can be recorded simultaneously from up to 60 channels (samplingfrequency up to 40 kHz) under sterile conditions. The data can beanalyzed off-line with a customized toolbox programmed for MATLAB (TheMathworks, Natick, Mass., USA) to detect field potentials.

Example 2

Extracellular Recording of Field Action Potentials (fAP) of PurifiedCardiomyocytes

In accordance with the methods described in international applicationsWO2004/113515 and WO2005/005621 incorporating the disclosure ofinternational applications WO02/051987 and WO99/01552 EGFP-positiveatrial and pacemaker-like cardiac cells can be derived from stem cellswhich have been genetically engineered with a recombinant GFP gene underthe control of selective promoters and carrying the puromycin resistancegene; see, e.g., Kolossov et al., FASEB J. 19 (2005), 577-579. Aftermass culture as, for example, described in international applicationWO2005/005621, comprising ten 12×12 cm² culture dishes withapproximately 4000 EBs/20 ml and medium exchange (IMDM+20% FCS) everysecond or third day, respectively, through cell screens with nylonmembrane, and selection with puromycin for 9-14 days the resultant EBsare transferred into 50 ml tubes and washed twice with PBS. Aftertreatment with trypsin for 10 minutes with twice pipetting with a 1 mlpipette almost perfect dissociation is achieved, approximately 1.5×10⁶green cells. The dissociated cells are plated on fibronectin-coatedplates and medium is exchanged the next day, i.e. on day 15 afterculture, thereby removing most of the cell debris and dead cells.

For plating on MEAs the resultant cardiac cells are washed twice withice cold PBS with Ca²⁺/Mg²⁺. Thereafter, the cells are incubated on icefor 30 minutes and washed twice with PBS w/o Ca²⁺/Mg²⁺. Trypsinizationis followed for 5 minutes at 37° C. resulting in approximately 8×10⁵EGFP-positive cells.

The cells are then plated on fibronectin-coated MEA (3.3×10⁵EGFP-positive cardiac cells). To this end, MEAs were plasma cleaned andcoated with bovine placental fibronectin for approximately 4 hours at 4°C. Residual fibronectin was sucked off and MEAs were dried on cleanbench.

Selected, dissociated and replated atrial and pacemaker cells onfibronectin-coated MEAs can be used for acute as well as longtermanalysis on a single level or in total by means of recording from thesubstrate integrated microelectrodes.

In another experiment ventricular cardiomyocytes were generated from EBsderived from mouse embryonic stem cell line D3 (see Doetschman et al.,1985, 1988), supra, which were trRnsfected with a construct comprisingthe puromycin resistance- and EGFP-expression cassettes under thecontrol of the murine MLC2v promoter (RPLC2v, gene bank Accession No.AF302688) based on the parental pIRES2-EGFP vector (Clontech).

For the setup of mass culture, these genetically modified ES cells aretrypsinized, seeded with 2×10⁵ cells/ml in KO-DMEM+15% SR and culturedin suspension on a shaker. On day 4 the resultant EBs are plated with500 to 2000 EBs per 15 cm cell culture dish with gelatin-coating (20dishes).

At day 11, 0.4 μl/ml puromycin is added to the cell culture dishes andon the following day the medium is exchanged and another 1 μl/mlpuromycin is added. At day 14 the cells are combined and trypsinized.From these cells 1.1×10⁵ EGFP-positive cells were plated on onefibronectin 6 cm dish with puromycin (0.4 μl/ml) resulting inapproximately 27 ventricular cardiomyocytes per EB. At day 17ventricular cells are trypsinized and plated on MEAs yielding 6×10⁴purified cells corresponding to approximately 15 ventricularcardiomyocytes/EB.

MEA recordings may be performed from the monolayer of purifiedventricular cardiomyocytes 1, 2 and 7 days after plating on MEA. Inaccordance with the present invention it could be shown that therecordings from selected electrodes reflect typical long field actionpotential durations (fAPD), as expected for ventricular cardiomyocytes.

As described above, purified ES cell-derived cardiomyocytes may also beseeded in lower density (5×10⁴ cells per MEA) and single cell analysismay be performed on these autonomously beating cells, for example bytransmission light and fluorescence picturing and recordings from singlecells on MEA electrodes.

Taken together it could be demonstrated in accordance with the presentinvention that ES cell-derived in vitro differentiated tissue and cellsin combination with micro-electrode array technique can be used forquantitative and qualitative toxicity testing and drug evaluation. Asdescribed in the examples, preferably either embryoid bodies may be usedor selected, dissociated and replated differentiated cells derivedthereof, depending on the parameters to be tested. In summary, anon-invasive, in vitro functional cell and tissue assay system isprovided.

It will be recognized that the compositions and procedures provided inthe description can be effectively modified by those skilled in the artwithout departing from the spirit of the invention embodied in theclaims that follow.

1-33. (canceled)
 34. An in vitro method for screening at least one testsubstance for an effect on an isolated population of cells, comprising:providing an electrode array comprising one or more isolated populationsof cells having been obtained by differentiating mouse or humanpluripotent stem cells, wherein the stem cells have been geneticallyaltered to comprise a selectable marker operably linked to a regulatorysequence specific for a first cell type, wherein the one or moreisolated population of cells have been differentiated and depleted ofundifferentiated cells and/or of undesired cell types by using aselection system that is lethal to the undesired cells and cell types byexpressing a selectable marker gene that renders cells of the first celltype resistant to the lethal effect, contacting the populations of cellsin an electrode array with the at least one test substance; andmeasuring the electrical activity of the contacted cells with theelectrode array and analyzing at least one parameter selected from thegroup consisting of: (i) Na+ channel activity, (ii) Ca2+/K+ channelactivity, (iii) K+ channel activity; (iv) amplitude and/or fieldpotential duration, (v) chronotropy, (vi) arrhythmia, (vii) pH-value,(viii) oxygen partial pressure, (ix) beating arrest, (x) contractility,(xi) analysis of AV-dissociation contractility, (xii) conductivityand/or impedance, (xiii) nitrous oxide-effects, or (ix) morphologicalchanges; and selecting a test substance that has an effect on at leastone parameter in the measuring step as compared to cells of the sametype which were not contacted with a test substance and wherein a changein said at least one parameter indicates that the test substance has aneffect on the populations of cells.
 35. The method of claim 34, whereinsaid cells are selected from the group consisting of neuronal cells,glial cells, cardiomyocytes, glucose-responsive insulin secretingpancreatic beta cells, hepatocytes, astrocytes, oligodendrocytes,chondrocytes, osteoblasts, retinal pigment epithelial cells,fibroblasts, keratinocytes, dendritic cells, hair follicle cells, renalduct epithelial cells, vascular endothelial cells, testicularprogenitors, smooth muscle cells, and skeletal muscle cell.
 36. Themethod of claim 34, wherein said pluripotent stem cells are embryonicstem (ES) cells.
 37. The method of claim 34, wherein the selectablemarker confers resistance to puromycin, neomycin, or hygromycin.
 38. Themethod of claim 34, wherein said electrode array comprises one or moreelectrodes.
 39. The method of claim 34, wherein said electrode array isa multi- or microelectrode array (MEA).
 40. The method of claim 38,wherein the electrode array is coated with fibronectin.
 41. The methodof claim 34, wherein the cells are cardiomyocytes.
 42. The method ofclaim 34, wherein the cells are neuronal cells.
 43. The method of claim34, wherein the cells are neural cells.
 44. The method of claim 34,wherein the cells are fibroblasts.
 45. The method of claim 34, whereinthe cells are keratinocytes.
 46. The method of claim 34, wherein thecells are smooth muscle cells.
 47. The method of claim 34, wherein thecells are skeletal muscle cells.
 48. The method of claim 41, wherein atleast one of the parameters analyzed is selected from the groupconsisting of: beating frequency, mean contractility, maximumcontraction and mean area of contraction is analyzed.
 49. The method ofclaim 34, wherein test substance is one of a collection of testsubstances.
 50. The method of claim 34, wherein said collection of testsubstances comprises about 10³ to about 10⁵ substances.
 51. The methodof claim 34, wherein three or more measurements are taken.
 52. Themethod of claim 34, further comprising removing the measured cells. 53.The method of claim 51, wherein said measurements are taken at differentpositions within the array.
 54. The method of claim 34, wherein saidselection step positively screens a test sub stance.
 55. The method ofclaim 34, wherein said selection step screens out a test substance. 56.The method of claim 34, wherein said selection step determines thetoxicity of the test substance on the cells.